Methods and compositions for treating obesity and/or diabetes and for identifying candidate treatment agents

ABSTRACT

Provided are methods and compositions for identifying candidate agents for treatment of obesity, liver disease, and/or diabetes. Such methods include, e.g., contacting a mammalian cell or cell population with a test agent, and measuring an expression level and/or activity level of ClpP in the mammalian cell or in cells of the cell population. Also provided are methods and compositions for treating an individual (e.g., one who is obese and/or has diabetes). Treatment methods include administering an inhibitor of ClpP to the individual (e.g., to prevent or reduce weight gain, to increase insulin sensitivity, and/or to increase glucose tolerance).

CROSS-REFERENCE

This application is a divisional of U.S. patent application Ser. No.16/129,296, filed Sep. 12, 2018, which application is acontinuation-in-part of International Application No. PCT/US2017/022584,filed Mar. 15, 2017, which application claims the benefit of priority toU.S. Provisional Patent Application No. 62/309,311, filed Mar. 16, 2016,which applications are incorporated herein by reference in theirentireties.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS A TEXT FILE

A Sequence Listing is provided herewith as a text file,“GLAD-408WO_SEQUENCE_LISTTNG_ST25.txt” created on Mar. 13, 2017 andhaving a size of 9 KB. The contents of the text file are incorporated byreference herein in their entirety.

INTRODUCTION

Mammalian ClpXP is a mitochondrial matrix protease complex that requiresATP to unfold and hydrolyze protein substrates. The ClpXP complexincludes a catalytic subunit (ClpP) and a regulatory subunit (ClpX). TheEscherichia coli ClpP homolog forms a barrel-shaped complex withhydrolytic active sites sequestered inside, while the Escherichia coliClpX homolog forms a hexamer ring that attaches to each end of thebarrel and is responsible for substrate recognition and unfolding.Prokaryotic ClpXP protease facilitates the degradation of damaged orunneeded polypeptides for protein quality control. Like E. coli ClpP,human ClpP also forms a chamber-like structure in the presence of ClpX.The physiological functions of ClpP in mitochondria, the prime energygenerators of mammalian cells that play critical roles in energyhomeostasis and metabolic regulation, are largely unknown.

There is a need in the art for methods and compositions for identifyingcandidate agents for treating diseases related to energy homeostasis andmetabolic regulation, such as obesity, liver disease, and/or diabetes.For example, there is a need in the art for methods and compositions foridentifying candidate agents for increasing insulin sensitivity,preventing and/or reducing weight gain, preventing and/or reducing fattissue, e.g., white adipose tissue, and the like. There is also a needin the art for methods and compositions for treating obesity, liverdisease, and/or diabetes (e.g., by increasing insulin sensitivity,preventing and/or reducing weight gain, preventing and/or reducing fattissue, e.g., white adipose tissue, and the like).

SUMMARY

Provided are methods and compositions for identifying candidate agentsfor treating obesity, liver disease, and/or diabetes (e.g., candidateagents for decreasing an amount of fat tissue in an individual,preventing or reducing weight gain of an individual, increasing insulinsensitivity of an individual, and/or increasing glucose tolerance of anindividual). In some embodiments of the present disclosure, such methodsinclude (a) contacting a mammalian cell or cell population (e.g., arodent cell, a mouse cell, a rat cell, a non-human primate cell, amonkey cell, a human cell, or a cell population thereof) with a testagent, and (b) measuring an expression level (e.g., protein and/or mRNA)and/or activity level of ClpP in the mammalian cell (the agent-contactedcell) or in cells of the agent-contacted cell population. It is thendetermined whether the test agent caused a reduction of ClpP expressionand/or activity. Those test agents that reduce ClpP expression (e.g.,protein and/or mRNA) and/or activity, can be identified as candidateagents for treatment. Thus, a compound is considered to be a. “testagent” prior to contact with a cell or cell population (e.g., in vitro,ex vivo, or in vivo), and if the compound (the test agent) reduces ClpPexpression (e.g., as can be shown by measuring levels of ClpP proteinand/or ClpP-encoding mRNA) and/or activity, it is then considered to bea “candidate agent” (e.g., a candidate agent for treatment, such as fortreating obesity, liver disease and/or diabetes). Thus, in some cases asubject method further includes a step (c): determining whether the testagent caused a reduction of ClpP expression (e.g., where a reduction ofClpP expression is indicative that the test agent is a candidate agentfor treatment) and/or activity. In some cases, a subject method includeseither: (step c) determining that the test agent caused a decrease inthe expression level and/or activity level of ClpP (e.g., relative to areference value, e.g., an expression level and/or activity level of ClpPprior to contact with the test agent, an expression level and/oractivity level of ClpP after contact with a control agent that is knownnot to reduce ClpP expression, and the like), and identifying the testagent as a candidate agent for treating obesity, liver disease, and/ordiabetes, or (step d) determining that the test agent did not cause adecrease in the expression level and/or activity level of ClpP (e.g.,relative to the reference value).

In some cases, the test agent is a small molecule or a polypeptide. Insome cases, the expression level (of ClpP) is an RNA expression leveland the measuring includes, e.g., the use of quantitative RT-PCR, amicroarray, or RNA sequencing. In some cases, the expression level (ofClpP) is a protein expression level and the measuring includes detectingClpP protein (e.g., using an anti-ClpP antibody, mass spectrometry,and/or an enzyme-linked immunosorbent assay (ELISA) assay). In somecases, the method includes screening a plurality of test agents toidentify one or more candidate agents for treating obesity, liverdisease, and/or diabetes.

In some cases, the mammalian cell (the target cell to be contacted withthe test agent) is a liver cell (a hepatocyte). In some cases, thecontacting is in vitro (e.g., the mammalian cell is in vitro). In somecases, the contacting is ex vivo (e.g., the mammalian cell is ex vivo).In some cases, the contacting is in vivo (e.g., the mammalian cell is invivo). In some cases, the contacting includes administering the testagent to a mouse. In some cases, the method includes a step of measuringan expression level and/or activity level of ClpP in the mouse prior tothe contacting of step (a) in order to obtain the reference value. Insome cases, the method includes a step of generating a report that thetest agent is a candidate agent for treating obesity, liver disease,and/or diabetes.

In some cases, a subject method includes, after determining that thetest agent is a candidate agent for treatment, a step of administeringthe identified candidate agent to an individual that has obesity, liverdisease, and/or diabetes (e.g., as a treatment or as a way to testwhether the candidate agent causes a desired outcome). In some cases,the individual is a mouse, a non-human primate, or a human. In somecases, the method includes, after administering the identified candidateagent to the individual, measuring one or more features of theindividual selected from: insulin sensitivity; blood glucose level;glucose tolerance; body fat mass; an amount of fat tissue; an amount ofwhite adipose tissue; percent fat mass; body weight; visceral adiposeadipocyte size; plasma leptin level; growth hormone level; basal energyexpenditure; a level of phosphorylated AKT (p-AKT) in muscles and/orfibroblasts; percent lean mass; mitochondrial number in hepatocytes;mitochondrial mass in hepatocytes; mitochondrial morphology inhepatocytes; fibroblast respiratory capacity; fibroblast maximal oxygenconsumption rate (OCR); and fibroblast resistance to H₂O₂-inducedcytotoxicity.

Also provided are methods and compositions for treating an individual(e.g., one who is obese and/or has diabetes or who has been diagnosed asbeing obese and/or having diabetes). Treatment methods includeadministering an inhibitor of ClpP (e.g., an agent that reduces theamount and/or activity of ClpP protein) to the individual in an amounteffective for decreasing an amount of fat tissue in the individual,preventing or reducing weight gain of the individual, increasing insulinsensitivity of the individual, and/or increasing glucose tolerance ofthe individual. In some cases, the inhibitor of ClpP is a smallmolecule, e.g., any small molecule ClpP inhibitor described herein,e.g., a β-Lactone, such as any β-Lactone molecule described herein. Insome cases, the small molecule is(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one. Insome cases, the inhibitor of ClpP is an RNAi agent or a gene editingagent that targets ClpP (e.g., specifically reduces the expression levelof the ClpP protein). In some cases, the inhibitor of ClpP isadministered such that reduction of ClpP expression is substantiallyliver-specific, and the amount administered is effective for increasinginsulin sensitivity of the individual. In some cases, the inhibitor ofClpP is delivered directly to the individual's liver, and the amountadministered is effective for increasing insulin sensitivity of theindividual. In some cases, the administering includes local injection.In some cases, a subject treatment method includes a step of measuringinsulin sensitivity of the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is best understood from the following detailed descriptionwhen read in conjunction with the accompanying drawings. It isemphasized that, according to common practice, the various features ofthe drawings are not to-scale. On the contrary, the dimensions of thevarious features are arbitrarily expanded or reduced for clarity.Included in the drawings are the following figures.

FIG. 1 (panels a-m) provides data showing that ClpP^(−/−) mice had lessbody fat and a resistance to high-fat diet-induced obesity. FIG. 1(panel a) provides images showing that ClpP^(−/−) mice were smaller andhad lower body fat. FIG. 1 (panel b) provides graphs showing that bodyweights of CdpP^(−/−) mice were lower than those of wild-type (WT) andClpP^(+/−) littermates (n=9 for WT female, n=10 for ClpP^(+/−) female,n=13 for ClpP^(−/−) female, n=13 for WT male, n=13 for ClpP^(−/−) male,n=10 for ClpP^(−/−) male). FIG. 1 (panel c) provides a graph showingthat new-born ClpP^(−/−) pups (postnatal day 1) had similar body weightsto WT and Clp^(−/−) pups (n=5 for WT, n=7 for ClpP^(+/−), n=6 forClpP^(−/−)). FIG. 1 (panel d), FIG. 1 (panel e) provide graphs depictingthe fat mass and fat content of 5-month-old male mice (n=7), FIG. 1(panel f), FIG. 1 (panel g) provide graphs depicting lean mass and leancontent of 5-month-old male mice (n=7). FIG. 1 (panel h), FIG. 1 (paneli) provide data depicting brown adipose tissue mass and content of 8month-old male mice (n=7 for WT and Clp^(+/−), n=4 for ClpP^(−/−). FIG.1 (panel j) provides a graph depicting body weight gain of 8-month-oldmale mice on a high-fat diet (HFD) (n=8 for WT and ClpP^(+/−), n=7 forClpP^(−/−)). FIG. 1 (panel k), FIG. 1 (panel l) provide graphs of fatmass and content of 8-month-old male mice after 40 days on HFD (n=8 forWT, n=7 for ClpP^(−/−) and ClpP^(−/−)). FIG. 1 (panel m) provides ahistogram of adipocyte size distribution of WT, ClpP^(+/−), andClpP^(−/−) mice. Data are mean±SD. *P<0.05, **P<0.01, ***P<0.005 versusWT.

FIG. 2 (panels a-i) provide data showing that ClpP^(−/−) mice hadaltered whole-body energy expenditure. FIG. 2 (panel a), FIG. 2 (panelh) provide graphs depicting food intake of 7-8-month-old male mice (n=12for WT and ClpP^(−/−), n=15 for ClpP^(+/−)). FIG. 2 (panel c) provides atimeline of fasting-refeeding experiments. FIG. 2 (panel d), FIG. 2(panel e) provide graphs depicting body weight loss of 7-8-month-oldmale mice after a 24-h fast (n=14 for WT and ClpP^(−/−), n=15 forClpP^(+/−)). FIG. 2 (panel f) provides graphs depicting food intake of7-8-month-old male mice during 0-8 hour or 8-24 hour refeeding periods(n=3 for WT and ClpP^(−/−), n=4 for ClpP^(+/−)). FIG. 2 (panel g)provides graphs depicting the percentage body weight gain of7-8-month-old male mice after 8 hour or 24 hour refeeding (n=14 for WTand ClpP^(−/−), n=15 for ClpP^(+/−)). FIG. 2 (panel h), FIG. 2 (panel i)provide graphs depicting body temperature of 8-month-old male mice aftera 16-h fast or during free feeding (n=13 for WT and ClpP^(+/−), n=14 forClpP^(−/−)). Data are mean±SD. *P<0.05, **P<0.01, ***P<0.005 versus WT.

FIG. 3 (panels a-o) provide data showing that ClpP^(−/−) mice hadincreased insulin sensitivity. FIG. 3 (panels a-c) provide graphsdepicting blood glucose and plasma insulin levels of 8-month-old malemice on chow diet (n=8 for each group). FIG. 3 (panel d), FIG. 3 (panele) provide graphs depicting blood glucose and plasma insulin levels of8-month-old male mice on a high-fat diet (n=5 for each group). FIG. 3(panel 0 provides a graph depicting glucose tolerance curve of6-10-month-old male mice (n=9 for each group). FIG. 3 (panel g) providesgraphs depicting plasma insulin levels of 6-10-month-old male mice afterglucose challenge (n=5 for each group). FIG. 3 (panel h) provides agraph depicting an insulin tolerance curve of 12-month-old male mice(n=7 for each group). FIG. 3 (panel i) provides a graph depicting apyruvate tolerance curve of 14-month-old male mice (n=5 for WT andClpP^(−/−), n=7 for ClpP^(−/−)). FIG. 3 (panel j), FIG. 3 (panel k)provide a representative image (j) and quantification (k) of pAKTimmunoblot of mouse fibroblasts from WT, ClpP^(−/−), or ClpP^(−/−) mice.FIG. 3 (panel l) provides a graph depicting pAKT levels in gastrocnemiusmuscles of 10-month-old male mice (n=3 for each group). FIG. 3 (panel m)provides a graph depicting pAKT levels in mouse fibroblasts after IGFtreatment (n=6 per dose for each group). FIG. 3 (panel n) provides agraph depicting blood glucose levels of 3-7-month-old db/db mice withdifferent ClpP genotypes after a 4-h fast (n=8 for WT, n=7 forClpP^(+/−), n=6 for ClpP^(−/−)). FIG. 3 (panel o) provides a glucosetolerance curve of 3-7-month-cold db/db mice with different ClpPgenotypes (n=8 for WT, n=7 for ClpP^(+/−), n=6 for ClpP^(−/−)). Data aremean±SD. *P<0.05, **P<0.01,***P<0.005 versus WT.

FIG. 4 (panels a-l) provide data showing that ClpP^(−/−) mice hadincreased mitochondrial chaperone levels. FIG. 4 (panels a-h) providerepresentative western blots and quantification of TRAP1, Grp75, LRPPRC,and ClpX in lysates from various organs of mice (n=3 for each group).FIG. 4 (panel i), FIG. 4 (panel j) provide representative western blots(i) and quantification (j) of ClpX, TRAP1, LRPPRC, and OAT in lysates ofWT or ClpP^(−/−) mouse fibroblasts. FIG. 4 (panel FIG. 4 (panel l)provide representative western blots (k) and quantification (1) of ClpX,LRPPRC, and OAT in ClpP^(−/−) fibroblasts transfected with an emptyvector (control) or a mouse ClpP cDNA construct. Data are mean±SD.*P<0.05, **P<0.01, ***P<0005 versus WT.

FIG. 5 (panels a-j) provide data showing that ClpP^(−/−) mice hadincreased mitochondrial numbers, improved mitochondrial function, andenhanced anti-oxidative stress capability. FIG. 5 (panel a) providesrepresentative electron microscopic image of mitochondria in WT andClpP^(−/−) mouse hepatocytes. Scale bars are 2 μm. FIG. 5 (panel b),FIG. 5 (panel c) provide graphs of mitochondrial number (b) and mass (c,measured by mitochondrial area) increased in ClpP^(−/−) mousehepatocytes. The mitochondrial numbers were counted for each randomfield at ×13,600 magnification (n=17 for WT, n=12 for ClpP^(−/−)). Thetotal mitochondrial area per field was measured by Image J at ×13,600magnification (n=17 for WT, n=12 for ClpP^(−/−)). FIG. 5 (panel d)provides an oxygen consumption rate (OCR) curve of fibroblasts fromClpP^(−/−) and WT mice under basal and uncoupling conditions (n=12 foreach group), FIG. 5 (panel e) provides a graph showing that fibroblastsfrom ClpP^(−/−) mice were resistant to H₂O₂-induced cell death comparedwith fibroblasts from WT mice (n=8 for each group). FIG. 5 (panel l)provides a graph showing that overexpression of mouse ClpP^(−/−)decreased the respiratory capacity of fibroblasts (n=12 for each group).FIG. 5 (panel g) provides a graph showing that overexpression of mouseClpP abolished the H₂O₂ resistance of ClpP^(−/−) fibroblasts (n=8 foreach group). FIG. 5 (panel h), FIG. 5 (panel i) provide graphs showingthat lentiviral shRNA-mediated knockdown of TRAP1 or Grp75 lowered theresistance of ClpP^(−/−) fibroblasts to H₂O₂ cytotoxicity (n=8 for eachgroup). FIG. 5 (panel j) provides a graph showing the effects oflentiviral shRNA-mediated knockdown of different mitochondrial proteinson the maximum respiration capacity of ClpP^(−/−) fibroblasts (n=9 foreach group). Data are mean±SD. *P<0.01, **P<0.01, ***P<0.005 versus WT(b-e) or ClpP^(−/−) (f-j).

FIG. 6 (panels a-i) provide data showing that AAV-Cre-mediated knockdownof ClpP in livers increased insulin sensitivity in ClpP-cKO mice. 3-5month-old ClpP-cKO mice were injected with AAV-CMV-Cre (Cre group, n=11)or control AAV (Con group, n=10) through tail vein at a dose of 5×10⁹gc/gram. FIG. 6 (panel a) provides images of ClpP immunostaining,showing a significant reduction of ClpP in livers of AAV-Cre-injectedClpP-cKO mice compared to control AAV-injected mice. Scale bars are 50μm. FIG. 6 (panels b-d) provide graphs of body weights (b and c) ofClpP-cKO mice before and 3 weeks after AAV injection. The body weightgain of the AAV-Cre-injected mice was significant lower than that ofcontrol AAV-injected mice (d). FIG. 6 (panel e) provides a graph ofplasma insulin levels of ClpP-cKO mice before and 3 weeks after AAVinjection. FIG. 6 (panel 0 provides a graph of blood glucose levels ofClpP-cKO mice before and 3 weeks after AAV injection. FIG. 6 (panel g)provides a glucose tolerance curve of ClpP-cKO mice 4 weeks after AAVinjection. FIG. 6 (panel h), provides a glucose tolerance curve ofClpP-CKO mice injected with AAV-Cre or control AAV and on HFD for 2weeks. FIG. 6 (panel i) provides a graph of data collected after HFD wasgiven to mice 6 weeks after AAV injection. No differences of body weightgain were detected after 2 or 4 weeks on HFD. Data are mean±SD. *P<0.01,***P<0.005 versus control.

FIG. 7 (panels a-i) provide data showing the generation of ClpP^(−/−)mice. FIG. 7 (panel a) provides a schematic of the gene-trappingstrategy used to generate ClpP^(−/−) mice. FIG. 7 (panels b-i) providewestern blots and quantification of ClpP protein levels in lysates ofthe liver, adipose tissue, muscle, and brain. Data are mean±SD.***P<0.005 versus WT.

FIG. 8 (panels a-b) provide data showing that ClpP^(−/−) mice havenormal histology of the liver and muscle. FIG. 8 (panel a), FIG. 8(panel b) provide representative H&E-stained images of the liver (a) andgastrocnemius muscle (b) WT, ClpP^(−/−), and ClpP^(−/−) mice. Scale barsare 50 μm.

FIG. 9 (panels a-j) provide data showing that ClpP^(−/−) mice havenormal neurological profile. FIG. 9 (panels a-e) provide graphs of datacollected from performing the grip test, incline test, tail suspensetest, and rotarod test of 8-month-old male mice (n=8 for each group).FIG. 9 (panel f) provides a graph from the elevated plus maze testperformed with 8-9-month-old male mice (n=8 for each group). FIG. 9(panels g-j) provide graphs from the Morris water maze test performedwith 8-9-month-old male and female mice (n=8 for each group) (—♦— WT,—≡— ClpP^(−/−), —▴— ClpP^(−/−)). H, hidden trial; V, visible trial. Dataare mean±SEM.

FIG. 10 (panels a-d) provide data showing that ClpP^(−/−) mice havesmaller adipocytes. FIG. 10 (panels a-b) provide representative imagesof H&E-stained adipose tissues from WT, ClpP^(+/−), and ClpP^(−/−) mice.

FIG. 11 (panels a-b) provide data showing that ClpP^(−/−) mice havenormal histology of brown adipose tissue and pancreas. FIG. 11 (panela), FIG. 11 (panel b) provide representative H&E-stained images of thebrown adipose tissue (a) and pancreas (b) in WT, ClpP^(+/−), andClpP^(−/−) mice. Scale bars are 100 μm,

FIG. 12 (panels a-d) provide data showing that ClpP^(−/−) mice areresistant to high-fat diet (HFD)-induced obesity. FIG. 12 (panel a),FIG. 12 (panel b) provide graphs of body weight gain of 8-month-old malemice after being on an HFD for 10 days and 20 days (n 8 for WT andClpP^(+/−), n=7 for ClpP). FIG. 12 (panel c), FIG. 12 (panel d) providegraphs of the lean body mass of 8-month-old male mice after being on theHFD for 40 days (n=6 for WT, n=7 for ClpP^(+/−) and ClpP^(−/−)). Dataare mean±SD. **P<0.01, ***P<0.005 versus WT.

FIG. 13 (panels a-b) provide data showing that ClpP^(−/−) mice havenormal locomotor activities. FIG. 13 (panel a), FIG. 13 (panel b)provide graphs depicting the total movement and the ratio of center tototal movement for 8-10-month-old male mice during an open field test(n=10 for WT, n=12 for ClpP^(+/−) and ClpP^(−/−)). Data are mean±SEM.

FIG. 14 (panels a-c) provide data related to the effects of knocking outClpP on obesity and insulin resistance in db/db mice. FIG. 14 (panel a)provides a graph of the body weight of 4-8-month-old db/db mice withdifferent ClpP genotypes (n=10 for WT, n=17 for ClpP^(+/−), n=8 forClpP^(−/−)), FIG. 14 (panel b), FIG. 14 (panel c) provide graphsdepicting the levels of blood glucose and plasma insulin of3-7-month-old db/db mice with different ClpP genotypes after a 16-h fast(n=8 for WT, n=7 for ClpP^(+/−), n=6 for ClpP^(−/−)). Data are mean SD.*P<0.05 versus WT.

FIG. 15 (panels a-c) provide representative images from comparative 2Dfluorescence difference gel electrophoresis (2D-DIGE) on differentorgans. FIG. 15 (panel a) provides an image of 2D-DIGE profiles of WTversus ClpP^(−/−) or WT versus ClpP^(+/−) livers. FIG. 15 (panel b)provides an image of 2D-DIGE profiles of WT versus ClpP^(−/−) or WTversus ClpP^(+/−) muscles. FIG. 15 (panel c) provides an image of2D-DIGE profiles of WT versus ClpP^(−/−) or WT versus ClpP^(+/−)hippocampi. Green, WT; red, ClpP^(−/−) or ClpP^(−/−).

FIG. 16 (panels a-b) provide data showing that the absence of ClpPincreases the levels of many mitochondrial proteins in various organs.FIG. 16 (panel a), FIG. 16 (panel b) provide Western blots andquantification of OAT, LonP, SDH2, ATP6V1A, VDAC, Hsp60, CPS1, Hsp70,Grp78, and Hsp60 in lysates of different oceans. Data are mean±SD.*P<0.05, **P 0.01, ***P<0.005 versus WT.

FIG. 17 (panels a-d) provide data showing that knocking out ClpP alteredmitochondrial numbers and morphology in mouse hepatocytes, FIG. 17(panel a), FIG. 17 (panel b) provide representative electron microscopicimage of mitochondria in WT (a) and ClpP^(−/−) (b) mouse hepatocytes.Scale bars are 2 μm. FIG. 17 (panel c) provides a histogram ofmitochondrial size distribution in WT and ClpP^(−/−) mouse hepatocytes(n=507 for WT, n=529 for ClpP^(−/−)). FIG. 17 (panel d) provides ahistogram of mitochondrial roundness distribution in WT and ClpP^(−/−)mouse hepatocytes (n=507 for WT, n=529 for ClpP^(−/−)). The roundnesswas defined as 4×(area)/(π×(major axis)²) and measured via Image J at×13,600 magnification.

FIG. 18 (panels a-f) provide data related to knocking down ClpPtargeting proteins in ClpP^(−/−) fibroblasts and a cell viability assay.FIG. 18 (panels a-d) provide representative western blots andquantifications of LRPPRC, TRAP1, Grp75, and ClpX protein levels inClpP^(−/−) fibroblasts treated by different lentiviral shRNAs for 48hours. FIG. 18 (panel e) provides a graph depicting cell viability inresponse to H₂O₂ treatment of ClpP^(−/−) fibroblasts treated with LRPPRClentiviral shRNAs (n=8 for each group) (—♦— ClpP^(−/−)+LRPPRC-shRNA).FIG. 18 (panel f) provides a graph depicting cell viability in responseto H₂O₂ treatment of ClpP^(−/−) fibroblasts treated with ClpX lentiviralshRNAs (n=8 for each group) (—♦— ClpP⁺, —▪— ClpP^(−/−)+ClpX-shRNA). Dataare mean±SD. **P<0.01, ***P<0.005 versus ClpP^(−/−).

FIG. 19 (panels a-f) provide data showing that adipocyte-specificknockout of ClpP did not affect body weight, blood glucose levels, andinsulin sensitivity in mice. FIG. 19 (panels a-b) provide a western blotof ClpP levels in adipose tissues of adipocyte-specific ClpP-cKO mice,and a graph of the data. FIG. 19 (panel e) provides a graph of bodyweights of 5-10-month-old adipocyte-specific ClpP-cKO mice compared tocontrols (no Crc littermates) (n=30 for no Cre, n=24 for Ad-Cre), FIG.19 (panel d) provides a graph of the blood glucose levels of5-10-month-old adipocyte-specific ClpP-cKO mice compared to controls (noCre littermates) (n=30 for no Cre, n=24 for Ad-Cre). FIG. 19 (panel e)provides a glucose tolerance curve of 3-6-month-old adipocyte-specificClpP-cKO mice compared to controls (no Cre littermates) (n=13 for noCre, n=11 for Ad-Cre) (—♦— no Crc, —▪— Ad-Cre). FIG. 19 (panel f)provides a graph of body weight gain of 9-12-month-oldadipocyte-specific ClpP-cKO mice in response to FWD (n=9 for no Cre,n=11 for Ad-Cre). FIG. 19 (panel g) provides a glucose tolerance curveof 9-12-month-old adipocyte-specific ClpP-cKO mice after 4 Week on HFD(n=9 for no Cre, n=11 for Ad-Cre). ***P<0.005 versus No Cre.

FIG. 20 provides a table of proteins differentially expressed in ClpP-KOtissues.

FIG. 21 provides a table of potential substrates of ClpXP.

FIG. 22 provides a table showing that the expression of potential ClpXPsubstrates were not upregulated at transcription levels, as determinedby microarray assays.

FIG. 23 provides a western blot analysis of ClpP substrates followingtreatment with the small molecule ClpP inhibitors A2-32-01, AV167, andAV179.

FIG. 24 provides graphs showing protein levels for ClpP substrates(normalized to GAPDH) following treatment with the small molecule ClpPinhibitors A2-32-01-AV167, and AV179.

FIG. 25 provides graphs showing protein levels for ClpP substrates(normalized to Actin) following treatment with the small molecule ClpPinhibitors A2-32-01. AV167, and AV179.

FIG. 26 provides graphs showing mRNA levels for ClpP substratesfollowing treatment with A2-32-01.

FIG. 27 provides an overview of the in vivo study design of Examples 9and 10.

FIG. 28 provides a graph showing body weight data from the first twodays of in vivo 12-32-01 treatment from Example 9.

FIG. 29 provides graphs showing the results of a glucose tolerance testfollowing in vivo administration of a ClpP inhibitor in a high fat diet(HFD)-induced obesity and diabetes mouse model.

FIG. 30 provides a schematic showing an experimental scheme of HFD,A2-32-01 treatment, and metabolic assays.

FIG. 31 provides a graph showing body weight change of HFD-fed WT micein response to A2-32-01 treatment (n=9 fir vehicle group, n=10 forA2-32-01 group) (left panel). FIG. 31 also provides a graph showing theresults of a glucose tolerance test showing increased insulinsensitivity in HFD-fed WT mice treated with A2-32-01 compared to thosetreated with vehicle (n=9 for vehicle group, n=10 for A2-32-01 group)(right panel).

FIG. 32 provides a graph showing ClpP activity was lower in livermitochondrion lysates from A2-32-01-treated mice compared to those fromvehicle-treated mice (n=8 for vehicle group, n=10 for A2-32-01 group)(left panel). FIG. 32 also provides a graph showing that the levels oftentative ClpP effectors, measured by western, were higher in livermitochondrion lysates from A2-32-01-treated mice compared to those fromvehicle-treated mice (n=8 for vehicle group, n=10 for A2-32-01 group)(right panel).

FIG. 33 provides representative H&E and oil red staining of liversections from vehicle or A2-32-01 treated WT mice on HFD. Themicrographs showed that A2-32-01 treatment lowered the lipidaccumulation in liver cells and restored normal morphology of livercells in WT mice on HFD, as compared to vehicle-treated WT mice on HFD.

FIG. 34, Panel A, provides a schematic showing an experimental scheme ofA2-32-01 treatment and metabolic assays in db/db mice. FIG. 34, Panel B,provides a graph showing body weight change of db/db mice in response toA2-32-01 treatment. (n=7 for vehicle group, n=9 for A2-32-01 group).

FIG. 35 provides, Panel C, provides a graph showing results of a glucosetolerance test showing increased insulin sensitivity in db/db micetreated with A2-32-01 compared with those treated with vehicle (n=7 forvehicle group, n=9 for A2-32-01 group). FIG. 35, Panel D, provides agraph showing that fasting blood glucose levels in db/db mice treatedwith A2-32-01 are significantly lower than those treated with vehicle(n=7 for vehicle group, n=9 for A2-32-01 group). FIG. 35, Panel E,provides a graph showing that ClpP activity was lower in livermitochondria lysates from db/db mice treated with A2-32-01 compared tothose treated with vehicle (n=7 for vehicle group, n=9 for A2-32-01group).

FIG. 36, panels F-I, provide representative H&E (Panels F, H) and oilred staining (Panels G, I) of liver sections from vehicle or A2-32-01treated db/dh mice. The micrographs showed that A2-32-01 treatmentlowered the lipid accumulation in liver cells and restored normalmorphology of liver cells in db/db mice, as compared to vehicle-treateddb/db mice.

DETAILED DESCRIPTION

Provided are methods and compositions for identifying candidate agentsfor the treatment of obesity, liver disease, and/or diabetes. In someembodiments, such methods include (a) contacting a mammalian cell orcell population with a test agent, and (h) measuring an expression level(e.g., protein and/or mRNA) and/or activity level of ClpP in themammalian cell (the agent-contacted cell) or in cells of theagent-contacted cell population. If the compound (the test agent)reduces ClpP expression (e.g., as can be show by measuring levels ofClpP protein and/or ClpP-encoding mRNA) and/or activity level, it isthen considered to be a “candidate agent” (e.g., a candidate agent fortreatment). Also provided are methods and compositions for treating anindividual (e.g., one who is obese and/or has diabetes or who has beendiagnosed as such). Treatment methods include administering an inhibitorof ClpP (e.g., an RNAi agent or a gene editing agent that targets ClpP)to the individual (e.g., to prevent or reduce weight gain, to increaseinsulin sensitivity, and/or to increase glucose tolerance).

Before the present methods and compositions are described, it is to beunderstood that this invention is not limited to the particular methodsor compositions described, as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, some potential andpreferred methods and materials are now described. All publicationsmentioned herein are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. It is understood that the present disclosuresupersedes any disclosure of an incorporated publication to the extentthere is a contradiction.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “acell” includes a plurality of such cells and reference to “the peptide”includes reference to one or more peptides and equivalents thereof,e.g., polypeptides, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application, Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

ClpP Protein

Mammalian ClpXP is a protein complex (a protease) that has a catalyticsubunit (the ClpP protein) and a regulatory subunit (the ClpX protein).As described herein, it has been discovered that the ClpP protein playsan important biological role in mitochondrial function in mammaliancells, and that altering the expression/function of ClpP haswide-ranging physiological consequences.

The wild type mouse and human ClpP protein amino acid sequences (andtheir encoding mRNAs) are depicted here.

Wild Type human ClpP (NP_006003.1)

*also known as “caseinolytic mitochondrial matrix peptidase proteolyticsubunit”, DFNB81, and PRLTS3

(SEQ ID NO: 1) MWPGILVGGARVASCRYPALGPRLAAHFPAQRPPQRTLQNGLALQRCLHATATRALPLIPIVVEQTGRGERAYDIYSRLLRERIVCVMGPIDDSVASLVIAQLLFLQSESNKKPIHMYINSPGGVVTAGLAIYDTMQYILNPICTWCVGQAASMGSLLLAAGTPGMRHSLPNSRIMIHQPSGGARGQATDIAIQAEEIMKLKKQLYNIYAKHTKQSLQVIESAMERDRYMSPMEAQEFGILDKVLVHPPQDGEDEPTLVQKEPVEAAPAAEPVPAST

DNA version of the mRNA Encoding Human ClpP (Above) (NM_006012.2)

*ORF is underlined

(SEQ ID NO: 3) CCTTAATGGCGCCCGCCCAGACTCCTGGAAGTGAGCGGCCTAGCGAGCGAGCTCCCAGGCGCAAAGCACGCCGGAAGCTGTAGTTCCGCCATCGGACGGAAGCCGACCGGGGCGTGCGGAGGGATGTGGCCCGGAATATTGGTAGGGGGGGCCCGGGTGGCGTCATGCAGGTACCCCGCGCTGGGGCCTCGCCTCGCCGCTCACTTTCCAGCGCAGCGGCCGCCGCAGCGGACACTCCAGAACGGCCTGGCCCTGCAGCGGTGCCTGCACGCGACGGCGACCCGGGCTCTCCCGCTCATTCCCATCGTGGTGGAGCAGACGGGTCGCGGCGAGCGCGCCTATGACATCTACTCGCGGCTGCTGCGGGAGCGCATCGTGTGCGTCATGGGCCCGATCGATGACAGCGTTGCCAGCCTTGTTATCGCACAGCTCCTCTTCCTGCAATCCGAGAGCAACAAGAAGCCCATCCACATGTACATCAACAGCCCTGGTGGTGTGGTGACCGCGGGCCTGGCCATCTACGACACGATGCAGTACATCCTCAACCCGATCTGCACCTGGTGCGTGGGCCAGGCCGCCAGCATGGGCTCCCTGCTTCTCGCCGCCGGCACCCCAGGCATGCGCCACTCGCTCCCCAACTCCCGTATCATGATCCACCAGCCCTCAGGAGGCGCCCGGGGCCAAGCCACAGACATTGCCATCCAGGCAGAGGAGATCATGAAGCTCAAGAAGCAGCTCTATAACATCTACGCCAAGCACACCAAACAGAGCCTGCAGGTGATCGAGTCCGCCATGGAGAGGGACCGCTACATGAGCCCCATGGAGGCCCAGGAGTTTGGCATCTTAGACAAGGTTCTGGTCCACCCTCCCCAGGACGGTGAGGATGAGCCCACGCTGGTGCAGAAGGAGCCTGTAGAAGCAGCGCCGGCAGCAGAACCTGTCCCAGCTAGCACCTGAGAGCTGGGCCTCCTCTCCAGAATCATGTGGAGGGGCCAGAGGCCTGCCAGACCCCCAGCTGGGCCCTGCTCACCCCTTGTTGCTGGGCTTGGAGGGGCCTCTTGAGGAACTTTTAATTTGCAGGGGTGCCCGCTATGGACGGGGCATTCCAGCTGAGACACTGTGATTTTAAATTAAATCTTTGTGGTCTTTGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

Wild Type Mouse ClpP (NP_059089.1)

*also known as “caseinolytic mitochondrial matrix peptidase proteolyticsubunit”, AU019820, and D17Wsu160e

(SEQ ID NO: 2) MWPRVLLGEARVAVDGCRALLSRLAVHFSPPWTAVSCSPLRRSLHGTATRAFPLIPIVVEQTGRGERAYDIYSRLLRERIVCVMGPIDDSVASLVIAQLLFLQSESNKKPIHMYINSPGGVVTAGLAIYDTMQYILNPICTWCVGQAASMGSLLLAAGSPGMRHSLPNSRIMIHQPSGGARGQATDIAIQAEEIMKLKKQLYNIYAKHTKQSLQVIESAMERDRYMSPMEAQEFGILDKVLVHPPQDGEDEPELV QKETATAPTDPPAPTST

DNA Version of the mRNA Encoding Mouse ClpP (Above) (NM_017393.2)

*ORF is underlined

(SEQ ID NO: 4) AGTGACTCCCGCAAAGCACGCCGGGTGTTGTAGTTCCGGAAGCCAAGCCGGAGTGCGCGTCGTCATGTGGCCCAGAGTGCTGCTGGGGGAGGCCCGGGTGGCTGTGGACGGATGTCGCGCTCTGTTGTCTCGCCTTGCCGTGCATTTCTCCCCGCCATGGACTGCTGTGAGCTGCTCACCCCTGCGGAGGAGCCTGCATGGAACTGCGACGCGAGCTTTCCCGCTCATCCCCATAGTGGTGGAGCAGACGGGTCGAGGCGAGCGCGCTTATGACATATACTCGAGGCTGTTGCGGGAACGCATCGTGTGCGTCATGGGCCCGATTGACGACAGTGTGGCCAGTCTGGTCATTGCCCAGCTGTTGTTCTTACAGTCTGAAAGCAACAAGAAGCCCATTCATATGTATATCAACAGCCCAGGTGGTGTGGTAACTGCGGGCCTGGCCATCTACGACACAATGCAGTACATCCTGAACCCCATCTGCACGTGGTGTGTTGGACAGGCTGCCAGCATGGGCTCCCTGCTCCTCGCTGCTGGCAGCCCGGGCATGCGCCATTCACTGCCCAATTCCAGAATCATGATCCACCAGCCCTCTGGAGGAGCCAGGGGCCAAGCCACAGACATCGCCATCCAGGCAGAGGAAATCATGAAGCTGAAAAAGCAGCTATACAACATCTACGCCAAACACACCAAGCAGAGCCTACAGGTGATCGAGTCAGCAATGGAGAGGGACCGCTACATGAGCCCCATGGAGGCCCAAGAGTTTGGCATCTTGGACAAGGTCTTGGTCCACCCACCTCAGGACGGGGAGGATGAGCCAGAACTGGTACAGAAGGAGACTGCCACAGCGCCGACGGATCCTCCTGCCCCGACAAGCACCTAAGGAGTGGAGACCAGACTGAAACTTCCTCTGCTGGGCCCAAGAACAACCCCTAGAGGAGATGTGGATTGAGGTTGCCCTCAGAGCAGGGCAGACTGCCTGAGACACTGTGATTTAAATTAAATCTTTGTAGTCTTTGTCCCATGTCTGAAGCACCTTCCATTACTTCTCCAAGACAGCAGGCCTCCTTCACCTTGACAAACCACTTCAGTAAGCAAACCCIGGCTCTCCIGGAACTAAACCAATCTAGCCTCAGACTCAGGTACCCACCTGCCTCACCTCCTGAGTGCTAGGATTAAAGGTGTACACCACCACACCTGACTTCAA

Screening Methods

Provided are screening methods for identifying candidate agents fortreating obesity, liver disease, and/or diabetes. By way of example,such candidate agents may include candidate agents for decreasing anamount of fat tissue in an individual, preventing or reducing weightgain of an individual, increasing insulin sensitivity of an individual,and/or increasing glucose tolerance of an individual. In someembodiments, screening methods provided herein include (a) contacting amammalian cell or cell population (e.g., a mouse cell, a rat cell, anon-human primate cell, a human cell, or a cell population thereof) witha test agent, and (b) measuring an expression level and/or activitylevel of ClpP (e.g., a protein and/or mRNA expression level or anactivity level of ClpP) in the mammalian cell (the agent-contacted cell)or in cells of the agent-contacted cell population to determine whetherthe test agent caused a reduction of ClpP expression and/or activitylevel in the cell or cells of the cell population.

Test agents that reduce ClpP expression (e.g., protein and/or mRNA)and/or activity level, can be identified as candidate agents for use inthe treatment of obesity, liver disease, and/or diabetes. Thus, acompound is considered to be a “test agent” prior to contact with a cellor cell population (e.g., in vitro, ex vivo, or in vivo), and if thecompound (the test agent) reduces ClpP expression (e.g., as can be showby measuring levels of ClpP protein and/or ClpP-encoding mRNA) and/oractivity level, it is then considered to be a “candidate agent” (e.g., acandidate agent for the treatment of diseases in which ClpP reduction isbeneficial, such as obesity, liver disease, and/or diabetes). Thus, theprovided screening methods can also be referred to as methods ofidentifying an inhibitor of ClpP, methods of identifying an agent thatreduces ClpP expression and/or activity level, methods of identifying aninhibitor of ClpP expression and/or activity level, and the like.

In some embodiments, a subject method (e.g., a method as describedabove) includes (a) contacting a mammalian cell (e.g., a mouse cell, arat cell, a non-human primate cell, a human cell) with a test agent; (b)measuring a decrease in the expression level and/or activity level ofClpP caused by the contacting step (e.g., relative to a reference value,e.g., an expression level and/or activity level of ClpP prior to contactwith the test agent, an expression level and/or activity level of ClpPafter contact with a control agent that is known not to reduce ClpPexpression, and the like); (c) determining that the test agent caused adecrease in the expression level and/or activity level relative to thereference value; and (d) identifying the test agent as a candidate agentfor treating obesity, liver disease, and/or diabetes.

In some cases, the contacting is in vitro (e.g., the cell is in cultureand is contacted in vitro). In some cases, the contacting is ex vivo(e.g., the cell is in culture and is a primary cell isolated from anindividual).

A subject screening method includes a step of contacting a cell (or cellpopulation) with a test agent. When a test agent reduces a ClpPexpression level (e.g., at the level of ClpP protein or mRNA encodingthe ClpP protein) and/or activity level, that agent is determined to bea candidate agent (e.g., for treating obesity, liver disease, and/ordiabetes). Thus, the contacting will generally be for a period of timesuch that a change in an expression level and/or activity level of ClpP(e.g., protein and/or mRNA expression level) can potentially bedetected. In other words, the period of contact will be for a suitableperiod of time after which one may reasonably expect that if a testagent is in fact a candidate agent for treatment, a change in ClpPexpression and/or activity level will be detectable, in some cases, thecontacting is for a period of time of 2 or more minutes (e.g., 5 or moreminutes, 10 or more minutes, 15 or more minutes, 30 or more minutes, 1or more hours, 2 or more hours, 5 or more hours, 6 or more hours, 12 ormore hours or more hours, etc.). In some cases, the contacting is for aperiod in a range of from 2 minutes to 48 hours (e.g., 5 minutes to 24hours, 5 minutes to 6 hours, 5 minutes to 2 hours, 15 minutes to 24hours, 15 minutes to 6 hours, 15 minutes to 2 hours, 1 hour to 24 hours,1 hour to 6 hours, or 1 hour to 2 hours).

In some cases, the contacting is in vivo. For example, in some cases, asubject method (e.g., a screening method) includes a step ofadministering an agent (e.g., a test agent or a candidate agent) to anindividual. A step of administering can serve a number of differentpurposes. For example, in some cases a subject method includes a step ofadministering a test agent to an individual (e.g., a mouse or a rat),and then measuring an expression level and/or activity level of ClpP inorder to determine whether the test agent is a candidate agent fortreatment. Such administration can be performed as part of the screenfor identifying those test agents that are candidate agents fortreatment.

As another example, in some cases a subject method (e.g., a screeningmethod) includes a step of administering a candidate agent (i.e., anagent that has already been determined to reduce an expression leveland/or activity level of ClpP, e.g., using a subject method andcontacting a cell in vitro, ex vivo, or in vivo). Such administrationcan be performed as a treatment step, or for example, in order todetermine whether the candidate agent causes a measurable change infeatures associated with obesity, liver disease, and/or diabetes (e.g.,decrease in an amount of fat tissue, prevention or reduction of weightgain, increase in insulin sensitivity, increase in glucose tolerance,and the like).

Target Cells

Any suitable mammalian cell or cell population can be used in theprovided screening methods as a target cell or cell population (i.e., acell or cell population that is contacted with a test agent). Forexample, in some cases, the target cell or cell population (the cell orcell population contacted with the test agent) is a mammalian cell, arodent cell (e.g., a mouse cell, a rat cell), a rabbit cell, a non-humanprimate cell, a human cell, etc. As noted above, the target cell or cellpopulation can be in vitro (e.g., from an establish cell line), ex vivo(e.g., primary cells), or in vivo. The target cell can be any suitabletype of cell (e.g., a stem cell, a progenitor cell, an adipocyte, aneuron, a cell of the pancreas, a fibroblast, an epithelial cells, akidney cell, an embryonic cell, and the like), and in some cases, thecontacted cell (the target cell) is a hepatocyte.

Examples of suitable mammalian cells for the subject screening methodsinclude, but are not limited to: monkey kidney CV1 line transformed bySV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293cells subcloned for growth in suspension culture, Graham et al., J. GenVirol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10);Chinese hamster ovary cells/-DHFR(CHO, Urlaub et al., Proc. Natl. Acad.Sci. USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol.Reprod, 23:243-251 (1980)); monkey kidney cells (CV1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TR1 cells (Mather et al.,Annals N.Y. Acad. Sci. 383:44-68 (1.982)); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Test Agents

A “test agent” (e.g., “test compound”) to be used in the providedscreening methods can be any suitable agent (e.g., including, but notlimited to, organic molecules, small molecules, polynucleotides, RNA,DNA, proteins, antibodies, peptides, lipids, carbohydrates, and thelike). A test agent can also be a mixture of chemical compounds. In somecases, an array of spatially localized test agents can be used (e.g., apeptide array, polynucleotide array, and/or combinatorial small moleculearray; where “array” refers to a collection of different molecularspecies immobilized on a surface). In some cases, a small moleculelibrary is screened (i.e., a test agent can be a member of a smallmolecule library). Test agents can be biological macromolecules, can bepart of a bacteriophage peptide display library, can be part of abacteriophage antibody (e.g., scFv) display library, a polysome peptidedisplay library, and the like. A test agent can be an extract made frombiological materials such as bacteria, plants, fungi, or animal (e.g.,mammalian) cells and/or tissues. As provided in the subject screeningmethods, test agents are evaluated for potential activity as agents fortreating obesity, liver disease, and/or diabetes (e.g., candidate agentsfor decreasing an amount of fat tissue in an individual, preventing orreducing weight gain of an individual, increasing insulin sensitivity ofan individual, and/or increasing glucose tolerance of an individual). Insome cases, a subject method is for screening a plurality of testagents. Test agents can be evaluated (screened) individually(sequentially), or in parallel.

In some cases, a test agent (e.g., a test compound) can have a formulaweight of less than 10,000 grams per mole (e.g., less than 5,000 gramsper mole, less than 1,000 grains per mole, or less than 500 grams permole). A test agent can be naturally occurring (e.g., an herb or anatural product), synthetic; or can include both natural and syntheticcomponents. Examples of small molecules include peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, and small molecules, such as organic or inorganic compounds,e.g., heterorganic or organometallic compounds.

ClpP Expression Level and/or Activity Level

In some cases, a subject screening method includes a step of measuring aClpP expression level and/or activity level. Because reduction of ClpPmRNA can result in reduced ClpP protein level, either assay (one tomeasure ClpP-encoding mRNA or one to measure ClpP protein) can be usedfor measuring an expression level. For example, in some cases, a testagent that reduces the level of ClpP protein in the target cell (or incells of the target cell population) will be identified as a candidateagent for use in treating obesity, liver disease, and/or diabetes. Insome cases, a test agent that reduces the level of ClpP-encoding mRNA inthe target cell (or in cells of the target cell population) will beidentified as a candidate agent for use in treating obesity, liverdisease; and/or diabetes.

The terms “assaying” and “measuring” are used herein to include thephysical steps of manipulating a biological sample (e.g., cell sample)to generate data related to the sample (e.g., measuring an expressionlevel and/or activity level in a biological sample). In practicing thesubject methods, the expression level of a ClpP expression product(e.g., mRNA or protein) and/or an activity level of ClpP can bemeasured. The expression level may be the expression level in a cell, ina population of cells, in a biological sample from an individual, andthe like. The expression level(s) and/or activity level(s) can bemeasured by any suitable method. For example, an RNA expression levelcan be measured by measuring the levels/amounts of one or more nucleicacid transcripts, e.g. mRNAs, of ClpP, Protein expression levels of ClpPcan be detected by measuring the levels/amounts of the ClpP protein.

“Measuring” can be used to determine whether the measured expressionlevel and/or activity level is less than, greater than. “less than orequal to”, or “greater than or equal to” a particular threshold, (thethreshold can be pre-determined or can be determined by assaying acontrol sample). On the other hand, “measuring to determine theexpression level” (and/or activity level) or simply “measuringexpression levels” (and/or activity levels) can mean determining aquantitative value (using any suitable metric) that represents the levelof expression (e.g., the amount of protein and/or RNA, e.g., mRNA) of aparticular expression product (e.g., a ClpP expression product) and/orthe activity level of ClpP. The level of expression and/or activity canbe expressed in arbitrary units associated with a particular assay(e.g., fluorescence units, e.g., mean fluorescence intensity (WO,threshold cycle (Ct), quantification cycle (CO, and the like), or can beexpressed as an absolute value with defined units (e.g., number of mRNAtranscripts, number of protein molecules, concentration of protein,amount of substrate cleaved, etc.).

An expression level and/or activity level can be a raw measured value,or can be a normalized and/or weighted value derived from the rawmeasured value. The terms “expression level” and “measured expressionlevel” are used herein to encompass raw measured values as well asvalues that have been manipulated in some way (e.g., normalized and/orweighted). In some cases, a normalized expression level and/or activitylevel is a measured expression level of an expression product and/or anactivity level from a sample where the raw measured value for theexpression product and/or activity level has been normalized. Forexample, the expression level of an expression product (e.g., an RNAencoding ClpP, a ClpP protein) can be compared to the expression levelof one or more other expression products (e.g., the expression level ofa housekeeping gene, the averaged expression levels of multiple genes,etc.) to derive a normalized value that represents a normalizedexpression level. Methods of normalization will be known to one ofordinary skill in the art and any suitable normalization method can beused. The specific metric (or units) chosen is not crucial as long asthe same units are used (or conversion to the same units is performed)when evaluating multiple markers and/or multiple biological samples(e.g., samples from multiple individuals or multiple samples from thesame individual).

A reduction of a ClpP expression level and/or activity level can bedetermined in a number of different ways. For example, in some cases, aClpP expression level and/or activity level is measured in a cell (or inan individual, e.g., in a biological sample from the individual) priorto and after contact with a test agent (or prior to and afteradministration of an agent to an individual), and a determination ismade as to whether a reduction was caused by the contacting (or theadministration). In some cases, some cells of a cell population are notcontacted with an agent (e.g., a test agent) and other cells of the cellpopulation are contacted with an agent (e.g., a test agent) and acomparison can be made among the contacted and non-contacted cells. Insome cases, some cells of a cell population are not contacted with anagent (e.g., a test agent) and other cells of the cell population arecontacted with a mock agent (e.g., an agent known not to cause a change)and a comparison can be made among the cells contacted with the testagent and those (control cells) contacted with the mock agent (controlagent).

Such comparisons can generally be referred to as comparing a measuredvalue to a reference value, or determining that an agent caused areduction in a ClpP expression level and/or activity level as comparedto a reference value. A reference value can be a level of ClpP (proteinand/or mRNA) and/or activity level of ClpP measured prior to contactwith an agent, a level of ClpP (protein and/or mRNA) and/or activitylevel of ClpP measured after contact with a mock agent (control agent),a level of ClpP (protein and/or mRNA) and/or activity level of ClpPmeasured in the absence of contact with an agent, etc. A reference valuecan also be in some cases a pre-determined (threshold) value.

In some cases, a subject method includes determining that a test agentor a candidate agent caused a decrease in the expression level and/oractivity level of ClpP. In some cases, the expression level and/oractivity level is reduced by 10% or more (e.g., 20% or more, 30% ormore, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more,or 90% or more) compared to a reference value (e.g., an activity leveland/or expression level, e.g., of ClpP protein and/or mRNA, prior tocontact with the agent; an activity level and/or expression level, e.g.,of ClpP protein and/or mRNA, in mock-treated cells or in mock-treatedcontrol individuals; etc.). In some cases, the measured expression leveland/or activity level is 95% of a reference value or less (e.g., 90% ofthe reference value or less, 85% of the reference value or less, 80% ofthe reference value or less, 75% of the reference value or less, 70% ofthe reference value or less, 65% of the reference value or less, 60% ofthe reference value or less, 55% of the reference value or less, 50% ofthe reference value or less, 45% of the reference value or less, 40% ofthe reference value or less, 35% of the reference value or less, 30% ofthe reference value or less, 25% of the reference value or less, 20% ofthe reference value or less, 15% of the reference value or less, 10% ofthe reference value or less, or 5% of the reference value or less). Insome cases, the reference value of the expression level and/or activitylevel of ClpP is 1.1-fold or more (e.g., 1.2-fold or more, 1.3-fold ormore, 1.4-fold or more, 1.5-fold or more, 2-fold or more, 2.5-fold ormore, 3-fold or more, 4-fold or more, 5-fold or more; 7.5-fold or more,or 10-fold or more) greater than the measured value.

Measuring RNA

An expression level of an expression product of ClpP may be measured bydetecting the amount or level of one or more RNA transcripts or afragment thereof encoded by the gene of interest (ClpP). Such detectionmay include detecting the level of one or more RNA transcripts or afragment thereof encoded by the ClpP gene in a cell extract, in a fixedcell, in a living cell, in a biological sample, etc. For measuring RNAlevels, the amount or level of an RNA in the sample is determined, e.g.,the expression level of an mRNA. In some instances, the expression levelof one or more additional RNAs may also be measured, and the level ofClpP RNA expression compared to the level of the one or more additionalRNAs to provide a normalized value for the ClpP expression level.

The expression level of nucleic acids in a sample (e.g., the expressionlevel of a ClpP mRNA) may be detected using any suitable protocol. Anumber of exemplary methods for measuring RNA (e.g., mRNA) expressionlevels in a sample are known by one of ordinary skill in the art, suchas those methods employed in the field of differential gene expressionanalysis, and any suitable method can be used. Exemplary methodsinclude, but are not limited to: hybridization-based methods (e.g.,Northern blotting, array hybridization (e.g., microarray); in situhybridization; in situ hybridization followed by FACS; and the like)(Parker & Barnes, Methods in Molecular Biology 106:247-283 (1999));RNAse protection assays (Hod, Biotechniques 13:852-854 (1992));PCR-based methods (e.g.; reverse transcription PCR (RT-PCR),quantitative RT-PCR (qRT-FCR), real-time RT-PCR, etc.)(Weis et al.,Trends in Genetics 8:263-264 (1992)); nucleic acid sequencing methods(e.g., Sanger sequencing, Next Generation sequencing (i.e., massiveparallel high throughput sequencing, e.g., Illumina's reversibleterminator method, Roche's pyrosequencing method (454), LifeTechnologies' sequencing by ligation (the SOLiD platform), LifeTechnologies' Ion Torrent platform, single molecule sequencing, etc.);nanopore based sequencing methods; and the like.

In some embodiments, the biological sample can be assayed directly. Insome embodiments, nucleic acid of the biological sample is amplified(e.g., by PCR) prior to assaying. As such, techniques such as PCR(Polymerase Chain Reaction), RT-PCR (reverse transcriptase PCR), qRT-PCR(quantitative RT-PCR, real time RT-PCR), etc. can be used prior to thehybridization methods and/or the sequencing methods discussed above.

Examples of some of the methods listed above are described in thefollowing references: Margulies et al (Nature 2005 437: 376-80); Ronaghiet al (Analytical Biochemistry 1996 242: 84-9); Shendure (Science 2005309: 1728); Imelfort et al (Brief Bioinform. 2009 10:609-18); Fox et al(Methods Mol Biol. 2009; 553:79-108); Appleby et al (Methods Mol Biol.2009; 513:19-39); Soni et al Clin Chem 53: 1996-2001 2007; and Morozova(Genomics. 2008 92:255-64), which are herein incorporated by referencefor the general descriptions of the methods and the particular steps ofthe methods, including starting products, reagents, and final productsfor each of the steps.

Measuring Protein

An expression level of an expression product of ClpP may be measured bydetecting the amount or level of one or more proteins (e.g., ClpP) or afragment thereof. Such detection may include detecting the level of aClpP protein or a fragment thereof in a cell extract, in a fixed cell,in a living cell, in a biological sample, etc. For measuring a proteinlevel, the amount or level of protein the sample (e.g., in a cell, in apopulation of cells, in a cell extract, etc.) is determined. In someinstances, the concentration of one or more additional proteins may alsobe measured, and the measured expression level compared to the level ofthe one or more additional proteins to provide a normalized value forthe measured expression level. In some embodiments, the measuredexpression level is a relative value calculated by comparing the levelof one protein relative to another protein. In other embodiments theconcentration is an absolute measurement (e.g., weight/volume orweight/weight).

The expression level of a protein (e.g., ClpP) may be measured bydetecting in a sample the amount or level of one or moreproteins/polypeptides or fragments thereof. The terms “polypeptide,”“peptide” and “protein” are used interchangeably herein to refer to apolymer of amino acid residues. “Polypeptide” refers to a polymer ofamino acids (amino acid sequence) and does not refer to a specificlength of the molecule. Thus peptides and oligopeptides are includedwithin the definition of polypeptide. In some cases, cells are removedfrom a biological sample (e.g., via centrifugation; via adhering cellsto a dish or to plastic, etc.) prior to measuring the expression level.In some cases, the intracellular protein level is measured by lysingcells of the sample to measure the level of protein in the cellularcontents. In some cases, a level of protein can be measured withoutdisrupting cell morphology (e.g., a protein level can be visualized,e.g., via fluorescent antibody staining, etc.) When protein levels areto be detected, any suitable protocol for measuring protein levels maybe employed. Examples of methods for assaying protein levels include butare not limited to antibody-based methods as well as methods that arenot antibody based. Examples of suitable methods include but are notlimited to: enzyme-linked immunosorbent assay (ELISA), massspectrometry, proteomic arrays, xMAP™ microsphere technology, flowcytometry, western blotting, immunofluorescence, andimmunohistochemistry.

Some protein detection methods are antibody-based methods. The term“antibody” is used in the broadest sense and specifically coversmonoclonal antibodies (including full length monoclonal antibodies),polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies); and antibody fragments so long as they exhibit the desiredbiological activity. Examples of antibody fragments include Fab, Fab′,Fab′-SH, F(ab′)₂, and Fv fragments; scFvs, diabodies; and multispecificor multivalent structures formed from antibody fragments.

ClpP Activity Level

As noted above, ClpP functions as part of a complex (ClpXP) thatincludes both ClpP and ClpX, and requires ATP to unfold and hydrolyzeprotein substrates. As such, any agent that disrupts the interactionbetween ClpP and ClpX, or that reduces the activity of the ClpP/ClpXcomplex (e.g., reduces the hydrolysis of protein substrates) can beconsidered an agent that reduces an activity level of ClpP. An activitylevel of ClpP can be measured using any suitable method. For example, anenzymatic assay can be used to measure ClpP activity. Parameters such asthe kinetics of hydrolysis of ATP by ClpXP and/or degradation ofsubstrate by ClpXP can be measured using any suitable method. Forexamples of various ways to measure ClpP activity level, see, e.g.,Burton et al., Protein Sci. 2003 May; 12(5):893-902; Joshi et al., NatStruct Mol Biol. 2004 May; 11(5):404-11; Kang et al., J Biol Chem. 2005Oct. 21; 280(42):35424-32; Baker et al., Biochim Biophys Acta. 2012January; 0823(1):15-28, and Al-Furoukh et al, (2014) PUS ONE 9(7):e103141, the disclosure of each of which are incorporated by referenceherein.

Furthermore, as demonstrated in the examples below, a decrease in ClpPactivity level (in this case caused by an absence of ClpP in knockoutmice) results in an increase in the amounts of the following proteins:TNF receptor-associated protein 1 (TRAP1) (mitochondrial Hsp90), heatshock protein family A (Hsp70) member 9 (Grp75) (mitochondrial Hsp70),leucine rich pentatricopeptide repeat containing (LRPPRC), caseinolyticmitochondrial matrix peptidase chaperone subunit (ClpX) (mitochondrialHsp100), ornithine aminotransferase (OAT), and Ion peptidase 1,initochondrial (LonP1) (a mitochondrial protease), As such, in somecases, measuring an activity level of ClpP can include measuring anamount of one or more (e.g., two or more, three or more, four or more, 5or more, or all 6) of the following proteins: TRAP1, Grp75, LRPPRC,ClpX, OAT, and LonP. For example, a decrease in ClpP activity can bedetected by measuring an increase in the levels of one or more (e.g.,two or more, three or more, four or more, 5 or more, or all 6) of:TRAP1, Grp75, LRPPRC, ClpX, OAT, and LonP, e.g., relative to a suitablecontrol, such as a level of one or more of TRAP1, Grp75, LRPPRC, ClpX,OAT, and LonP prior to contacting with a test agent as described herein.

Evaluation Steps

As described in the examples below, the inventors have discovered thatmice with decreased ClpP expression (due to knockout in the genome ofthe ClpP gene, designated ClpP ^((−/−))) exhibit a number of phenotypes,including but not limited to decreased: white adipose tissue, body fat(as measured by total body fat mass or body fat content (% fat mass)),body weight, visceral adipose adipocyte size, levels of plasma leptin,and blood glucose level (e.g., after fasting); and increased: insulinsensitivity, glucose tolerance, growth hormone levels, energyconsumption (e.g., increased basal energy expenditure), levels ofphosphorylated AKT (p-AKT) in muscles and/or fibroblasts, and leancontent (% lean mass). ClpP mice gained less weight and generated lessfat while consuming more food, and ClpP^(−/−) mice were resistant tohigh fat diet (HFD)-induced weight gain (e.g., the increase in fat massand body weight due to HFD was less than the increase seen in wild typecontrols).

In some cases, a subject method (e.g. a screening method or a treatmentmethod) includes, after administration of an agent to an individual(e.g., a candidate agent for treatment as identified with a subjectscreening method, an inhibitor of ClpP such as an RNAi agent or a genomeediting agent specific for ClpP, etc.), a step of measuring one or morefeatures of the individual (e.g., to verify that the agent produces adesired outcome). Suitable features that can be measured include, butare not limited to insulin sensitivity, blood glucose level, glucosetolerance, body fat mass, an amount of fat tissue, an amount of whiteadipose tissue, percent fat mass, body weight, visceral adiposeadipocyte size, plasma leptin level, growth hormone level, basal energyexpenditure, a level of phosphorylated AKT (p-AKT) in muscles and/orfibroblasts, percent lean mass, mitochondrial number in hepatocytes,mitochondrial mass in hepatocytes, mitochondrial morphology inhepatocytes, fibroblast respiratory capacity, fibroblast maximal oxygenconsumption rate (OCR), and fibroblast resistance to H₂O₂-inducedcytotoxicity. An evaluation step (e.g., a step of measuring one or moreof the above features), can be included as part of a subject screeningmethod (e.g., a method of identifying a candidate agent). An evaluationstep (e.g., a step of measuring one or more of the above features), canalso be included as part of a subject treatment method.

Generating a Report

In some cases, a subject method (e.g., any of the screening methodsdescribed above) includes a step of generating a report (e.g., a reportthat the test agent is a candidate agent for treating obesity, liverdisease, and/or diabetes). A “report,” as described herein, is anelectronic or tangible document which includes report elements thatprovide information of interest relating to the results and/orassessments of such results of a subject method (e.g., a screeningmethod). In some embodiments, a subject report includes a measuredexpression level and/or activity level as discussed in greater detailabove (e.g., a raw value, a normalized value, a normalized and weightedvalue, etc.) (e.g., an activity level of ClpP or an expression level ofa ClpP expression product, such as a ClpP protein and/or a ClpP-encodingmRNA). In some embodiments, a subject report includes a ClpP expressionlevel and/or activity level. In some cases, a subject report includes anassessment (e.g. a determination of whether one or more test agentscaused a reduction in a ClpP expression level and/or activity level).For example, a report can state whether a given test agent or list oftest agents caused a reduction in a ClpP expression level and/oractivity level (e.g., at the level of protein and/or mRNA), and/orwhether a given test agent or list of test agents is a candidate agentfor treatment.

Treatment Methods

Provided are methods that include administering to an individual aninhibitor of ClpP (such as an RNAi agent or gene editing agent specificfor ClpP), where the inhibitor of ClpP reduces an activity level and/orexpression level of ClpP (e.g., as can be detected at the level ofprotein and/or mRNA). Such methods can be referred to as methods ofreducing an expression level and/or activity level of ClpP, and/ormethods of treating an individual with obesity, liver disease, and/ordiabetes. A discussion of ClpP activity levels and expression levels(e.g., at the level of protein and/or mRNA), and methods for measuringsuch levels can be found elsewhere herein.

In some embodiments, such methods are methods of treating an individualwith obesity, liver disease, and/or diabetes, methods of increasingenergy output, methods of reducing an amount of adipose tissue (e.g.,white adipose tissue), methods of preventing or reducing weight gain,methods of increasing insulin sensitivity, and/or methods of increasingglucose tolerance. For example, in some cases, a subject method is amethod of increasing energy output, reducing an amount of adiposetissue, increasing insulin sensitivity, increasing glucose tolerance,and/or preventing or reducing weight gain, and the method includesadministering to the individual a ClpP inhibitor (such as an RNAi agentor gene editing agent specific for ClpP) that reduces the expressionlevel and/or activity level of ClpP. In some embodiments, a subjectmethod is a method of treating an individual with obesity, liverdisease, and/or diabetes, and the method includes administering to theindividual an inhibitor of ClpP (such as an RNAi agent or gene editingagent specific for ClpP) that reduces the expression level and/oractivity level of ClpP.

In some embodiments, a subject method is a method of administering aninhibitor of ClpP to an individual (e.g., an individual who has obesity,liver disease, and/or diabetes) in an amount effective for decreasing anamount of fat tissue in the individual, preventing or reducing weightgain of the individual, increasing insulin sensitivity of theindividual, and/or increasing glucose tolerance of the individual.

In some cases, any of the above subject treatment methods can include astep of measuring a ClpP expression level and/or activity level in abiological sample from the individual that is being treated (e.g., in ahepatocyte of/from the individual to whom a ClpP inhibitor wasadministered). In some cases, a subject treatment method includes a stepof obtaining a biological sample from the individual and measuring aClpP expression level and/or activity level of the sample. In somecases, such a step is performed before and after treatment (e.g., toverify that administration had the desired outcome), In some cases, aninhibitor of ClpP (e.g., an RNAi agent or a gene editing agent thattargets ClpP) is administered to an individual (e.g., an individual whois obese and/or has diabetes), in an amount effective for decreasing anamount of fat tissue in the individual, preventing or reducing weightgain of the individual, increasing insulin sensitivity of theindividual, and; or increasing glucose tolerance of the individual.

RNAi Agents and Genome Editing Agents

In some cases, an inhibitor of ClpP (e.g., an agent that reduces anexpression level and/or activity level of ClpP) is an RNAi agent or agenome editing agent that targets ClpP (e.g., is specific for ClpP). Theterm “RNAi agent” is used herein to mean any agent that can be used toinduce a gene specific RNA interference (RNAi) response in a cell.Suitable examples of RNAi agents include, but are not limited to shortinterfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), and microRNAs (miRNA). An RNAi agent (e.g., shRNA, siRNA, miRNA) specific forClpP is an agent that targets the LIANA encoding the ClpP protein. RNAiagents can readily be designed to specifically target any desired mRNA(e.g., one encoding ClpP) by choosing an appropriate nucleotidesequence.

Various RNAi agent designs (RNAi agents with various features) are knownin the art and any suitable RNAi agent that targets ClpP can be used.For example, various designs of RNAi agents (as well as methods of theirdelivery) can be found in numerous patents, including, but not limitedto U.S. Pat. Nos. 7,022,828; 7,176,304; 7,592,324; 7,667,028; 7,718,625;7,732,593; 7,772,203; 7,781,414; 7,807,650; 7,879,813; 7,892,793;7,910,722; 7,947,658; 7,973,019; 7,973,155; 7,981,446; 7,993,925;8,008,271; 8,008,468; 8,017,759; 8,034,922; 8,399,653; 8,415,319;8,426,675; 8,466,274; 8,524,679; 8,524,679; 8,569,065; 8,569,256;8,569,258; 9,233,102; 9,233,170; and 9,233,174; all of which areincorporated herein by reference. Where appropriate, an RNAi agent maybe provided in the form of a DNA encoding the agent (e.g., an expressionvector encoding a shRNA). shRNAs targeting the ClpP (accession no.NM_003321) coding sequence are provided for example in Cole et al.,2015, Cancer Cell 27, 864-876, the disclosure of which is incorporatedby reference herein in its entirety and for all purposes. These shRNAsinclude 5′-GCCCATCCACATGTACATCAA-3′ (SEQ ID NO:5);5′-CACGATGCAGTACATCCTCAA-3′ (SEQ NO:6); and 5′-GCTCAAGAAGCAGCTCTATAA-3′(SEQ ID NO:7).

The terms “genome editing agent” and “genome targeting composition” areused interchangeably herein to mean a composition that includes a genomeediting nuclease. In some embodiments, the genome editing nuclease bindsa native or endogenous recognition sequence. In some embodiments, thegenome editing nuclease is a modified endonuclease that binds anon-native or exogenous recognition sequence and does not bind a nativeor endogenous recognition sequence.

Examples of suitable genome editing nucleases include but are notlimited to zinc finger nucleases (ZFNs), TAL-effector DNA bindingdomain-nuclease fusion proteins (transcription activator-like effectornucleases (TALENs)), CRISPR/Cas endonucleases (e.g., class 2 CRISPR/Casendonucleases such as a type II, type V, or type VI CRISPR/Casendonucleases), and recombinases (e.g., Cre recombinase, Hinrecombinase, RecA, RAD51, Tre, FLP, and the like). Thus, in someembodiments, a genome editing agent is a composition that can includeone or more genome editing nucleases selected from: a ZFN, a TALEN, arecombinase (e.g., Cre recombinase, Hin recombinase. RecA, RAD51, ire,FLP, and the like), and a CRISPR/Cas endonuclease (e.g., a class 2CRISPR/Cas endonuclease such as a type II, type V, or type VI CRISPR/Casendonuclease).

Recombinases include but are not limited to Cre recombinase, Hinrecombinase, RecA, RAD51, Tre, and FLP.

Information related to class 2 type II CRISPR/Cas endonuclease Cas9proteins and Cas9 guide RNAs (as well as methods of their delivery) (aswell as information regarding requirements related to protospaceradjacent motif (PAM) sequences present in targeted nucleic acids) can befound in the art, for example, see Jinek et al., Science. 2012 Aug. 17;337(6090:816-21; Chylinski et al., RNA Biol. 2013 May; 10(5):726-37; Maet al., Biomed Res Int. 2013; 2013:270805; Hou et al., Proc Natl Acad.Sci USA. 2013 Sep. 24; 110(39):15644-9; Jinek et al., Elife. 2013;2:e00471; Pattanayak et al., Nat Biotechnol. 2013 September;31(9):839-43; Qi et al, Cell. 2013 Feb. 28; 152(5):1173-83; Wang et al.,Cell. 2013 May 9; 153(4):910-8; Auer et. al., Genome Res, 2013 Oct. 31;Chen et. al., Nucleic Acids Res. 2013 Nov. 1; 41(20):e19; Cheng et. al.,Cell Res. 2013 October; 23(10):1163-71; Cho et. al., Genetics. 2013November; 195(3):1177-80; DiCarlo et al., Nucleic Acids Res. 2013 April;41(7):4336-43; Dickinson et. al., Nat Methods. 2013 October;10(10):1028-34; Ebina et. al., Sci Rep. 2013; 3:2510; Fujii et, al,Nucleic Acids Res, November 1; 41(20):e187; Hu et. al., Cell Res. 2013November; 23(11):1322-5; Jiang et. al., Nucleic Acids Res. 2013 Nov. 1;41(20):e188; Larson et. al., Nat Protoc. 2013 November; 8(11):2180-96;Mali et. at., Nat Methods. 2013 October; 10(10):957-63; Nakayama et.al., Genesis. 2013 December; 51(12):835-43; Ran et. al., Nat Protoc.2013 November; 8(11):2281-308; Ran et. al., Cell. 2013 Sep. 12;154(6):1380-9; Upadhyay et. al., G3 (Bethesda). 2013 Dec. 9;3(12):2233-8; Walsh et. al., Proc Natl Acad Sci USA. 2013 Sep. 24;110(39):15514-5; Xie et. al., Mol Plant. 2013 Oct. 9; Yang et, al.,Cell. 2013 Sep. 12; 154(6):1370-9; Briner et al., Mol Cell. 2014 Oct.23; 56(2):333-9; and U.S. patents and patent applications: U.S. Pat.Nos. 8,906,616; 8,895,308; 8,889,418; 8,889,356; 8,871,445; 8,865,406;8,795,965; 8,771,945; 8,697,359; 20140068797; 20140170753; 20140179006;20140179770; 20140186843; 20140186919; 20140186958; 20140189896;20140227787; 20140234972; 20140242664; 20140242699; 20140242700;20140242702; 20140248702; 20140256046; 20140273037; 20140273226;20140273230; 20140273231; 20140273232; 20140273233; 20140273234;20140273235; 20140287938; 20140295556; 20140295557; 20140298547;20140304853; 20140309487; 20140310828; 20140310830; 20140315985;20140335063; 20140335620; 20140342456; 20140342457; 20140342458;20140349400; 20140349405; 20140356867; 20140356956; 20140356958;20140356959; 20140357523; 20140357530; 20140364333; and 20140377868; allof which are hereby incorporated by reference in their entirety.Examples and guidance related to type V or type VI CRISPR/Casendonucleases and guide RNAs (as well as information regardingrequirements related to protospacer adjacent motif (PAM) sequencespresent in targeted nucleic acids) can be found in the art, for example,see Zetsche et al, Cell. 2015 Oct. 22; 163(3):759-71; Makarova et al,Nat Rev Microbiol. 2015 November; 13(11):722-36; and Shmakov et al., MolCell. 2015 Nov. 5; 60(3):385-97.

Useful designer zinc finger modules include those that recognize variousGNN and ANN triplets (Dreier, et al., (2001) J Biol Chem 276:29466-78;Dreier, et al., (2000) J Mol Biol 303:489-502; Liu, et al., (2002) JBiol Chem 277:3850-6), as well as those that recognize various CNN orTNN triplets (Dreier, et al., (2005) J Biol. Chem 280:35588-97;Jamieson, et al., (2003) Nature Rev Drug Discov 2:361-8). See also,Durai, et al., (2005) Nucleic Acids Res 33:5978-90; Segal, (2002)Methods 26:76-83; Porteus and Carroll, (2005) Nat Biotechnol 23:967-73;Pabo, et al., (2001) Ann Rev Biochem 70:313-40; Wolfe, et al., (2000)Ann Rev Biophys Biomol Struct 29:183-212; Segal and Barbas, (2001) CurrOpin Biotechnol 12:632-7; Segal, et al., (2003) Biochemistry 42:2137-48;Beerli and Barbas, (2002) Nat Biotechnol 20:135-41; Carroll, et al.,(2006) Nature Protocols 1:1329; Ordiz, et al., (2002) Proc Natl Acad SciUSA 99:13290-5; Guan, et al., (2002) Proc Natl Acad Sci USA99:13296-301.

For more information on ZFNs and TALENs (as well as methods of theirdelivers), refer to Sanjana et al., Nat Protoc. 2012 Jan. 5; 7(1):171-92as well as international patent applications WO2002099084; WO00/42219;WO02/42459; WO2003062455; WO03/080809; WO05/014791; WO05/084190;WO08/021207; WO09/042186; WO09/054985; WO10/079430; and WO10/065123;U.S. Pat. Nos. 8,685,737; 6,140,466; 6,511,808; and 6,453,242; and USPatent Application Nos. 2011/0145940, 2003/0059767, and 2003/0108880;all of which are hereby incorporated by reference in their entirety.

Small Molecule ClpP Inhibitors

Small molecule inhibitors of ClpP are known Which may find use in one ormore of the screening or treatment methods described herein. For exampleβ-Lactones, such as(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one (alsoknown as A2-32-01 and referred to herein interchangeably as A2-32-01 andA2-32) have been identified as inhibitors of both bacterial andmammalian ClpP. See, e.g., Cole et al., 2015, Cancer Cell 27, 864-876,the disclosure of which is incorporated by reference herein in itsentirety and for all purposes.

Beta-lactones are described, for example, in U.S. Patent ApplicationPublication 2014/0243255, the disclosure of which is incorporated byreference herein in its entirety and for all purposes, and can include,but are not limited to, trans-beta-lactones, beta-propiolactone,saturated aliphatic beta-lactones, beta-butyrolactone,beta-isobutyrolactone, beta-valerolactone, beta-isovalerolactone,beta-n-caprolactone, alpha-ethylbeta-propiolactone,alpha-isopropyl-beta-propiolactone, alpha-butyl-beta-propiolactone,alpha, isopropyl-beta-propiolactone, beta isopropyl-beta-propiolactone,alpha-methyl-beta-butyrolactone,beta-ethyl-beta-butyrolactone-alpha-ethyl-beta-butyrolactone,alpha-methyl beta-propiolactone, lactones of betahydroxy-mono-carboxylicacids containing cycloalkyl, aryl and aralkyl substituents such asbetacyclohexyl-beta-propiolactone, beta-phenyl-betapropiolactone,alpha-phenyl-beta-propiolactone, beta-taenzyl-beta-propiolactone andderivatives thereof.

β-Lactones inhibitors of ClpP, which may be utilized in the context ofthe disclosed methods, include those described in U.S. PatentApplication Publication No. 2016/0221977, the disclosure of which isincorporated by reference herein in its entirely and for all purposes,Such β-Lactones inhibitors of ClpP include compounds of the followingstructures:

wherein R₂ is a single substitution of a hydrogen at any position on thebenzene ring where the substituted moiety is selected from the groupconsisting of alkyl, substituted alkyl, alkynyl, substituted alkyl,vinyl, nitro, halo (e.g., includes bromine, chlorine, fluorine andiodine), cyano, amyl, hetero aryl, alkoxy; and C_(n) is carbon and n isa number of from 1 to 5.

wherein R₁ is selected from the group consisting of alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, alkoxyalkyl, substituted alkoxyalkyl, NH2,NHR, NR2, mono- or polyhydroxy-substituted alkyl, aryl, substitutedheteroaryl, and substituted heteroaryl; R₂ is a single substitution of ahydrogen at any position on the benzene ring where the substitutedmoiety is selected from the group consisting of alkyl, substitutedalkyl, alkynyl, substituted alkyl, vinyl, nitro, halo, cyano, aryl,heteroaryl, alkoxy; and, C_(n) is carbon and n is a number of from 1 to5. Specific forms of the above structure are provided below:

Small molecule phenyl esters have also been shown to inhibit bacterialClpP. See, e.g., Hackl et al., J. Am. Chem. Soc., 137, 8475-8483 (2015),the disclosure of which is incorporated by reference herein in itsentirety and for all purposes. Such small molecule inhibitors include,e.g.:

In addition to the compounds described above, pharmaceuticallyacceptable salts and pro-drugs thereof may be utilized in the methodsdisclosed herein.

Agent Delivery

In some embodiments, a subject method (e.g., a screening method or atreatment method as described herein) includes a step of administeringan agent to an individual (e.g., a test agent, a candidate agent, a ClpPinhibitor that reduces an expression level and/or activity level ofClpP, e.g., an RNAi agent or a gene editing agent that specificallyreduces expression of ClpP, and the like). Depending on context, theindividual can be of any suitable species (e.g., a mammal, a rodent, amouse, a rat, a non-human primate, a human, etc.). For example, in somecases a test agent is administered to a mouse. In some cases, acandidate agent (e.g., as identified by a subject screening method) isadministered to a mouse, a rat, a non-human primate, or a human (e.g.,an individual with obesity, diabetes, reduced insulin sensitivity,reduced glucose tolerance, etc. in some cases, a ClpP inhibitor thatreduces an expression level and/or activity level of ClpP as describedherein is administered to a mouse, a rat, a non-human primate, or ahuman.

In some cases, an agent as described herein is administeredsystemically. In some cases, an agent as described herein isadministered locally (e.g., directly to a desired tissue such as theliver). In some cases, an agent as described herein is administered byparenteral, topical, intravenous, intratumoral, oral, subcutaneous,intraarterial, intracranial, intraperitoneal, intranasal orintramuscular means. A typical route of administration is intravenous orintratumoral, although other routes can be equally effective. In somecases, an agent as described herein is administered via injection. Insome cases, an agent as described herein is administered in atissue-specific manner (e.g., administration is directed to a specifictissue such as the liver).

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

In some embodiments, a single dose of a ClpP inhibitor is administered.In other embodiments, multiple doses of a ClpP inhibitor areadministered. Where multiple doses are administered over a period oftime, a ClpP inhibitor is administered twice daily (qid), daily (qd),every other day (god), every third day, three times per week (tiw), ortwice per week (biw) over a period of time. For example, a ClpPinhibitor is administered qid, qd, god, tiw, or biw over a period offrom one day to about 2 years or more. For example, a ClpP inhibitor isadministered at any of the aforementioned frequencies for one week, twoweeks, one month, two months, six months, one year, or two years, ormore, depending on various factors.

In some cases, a small molecule inhibitor of ClpP as described herein,e.g., a β-Lactone, such as(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one or apharmaceutically acceptable salt or pro-drug thereof is administered toa subject in need thereof, e.g., a subject as described herein, in anamount from about 50 mg/kg to about 1000 mg/kg per day, e.g., from about100 mg/kg to about 900 mg/kg per day, from about 200 mg/kg to about 800mg/kg per day, from about 300 mg/kg to about 700 mg/kg per day, or fromabout 400 mg/kg to about 600 mg/kg per day. In some cases, a smallmolecule inhibitor of ClpP as described herein, e.g., a β-Lactone, suchas (3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one ora pharmaceutically acceptable salt or pro-drug thereof is administeredto a subject as a once daily dose or a twice daily dose.

In some cases, a RNAi agent or genome editing agent is configured suchthat the reduction of ClpP expression brought about by the agent islimited to a particular tissue type (e.g., the liver). For example, thecomponents of an RNAi agent (e.g., shRNA, siRNA) or genome editing agent(CRISPR/Cas protein plus guide RNA) can be active only in a particulartissue, e.g., the components can be delivered to the particular tissue,the components can be operably linked to a tissue-specific promoter,etc. For example, as described in the examples below, delivery of theCre recombinase to the liver in mice that harbor a foxed allele of theClpP gene in their genome, recapitulates the increase in insulinsensitivity exhibited by ClpP knockout mice. Thus, reduction of ClpPexpression in the liver can result in an increase in insulin sensitivitywithout the potential side effects of reducing ClpP expressionthroughout the whole individual. In some cases, the inhibitor of ClpPthat reduces ClpP expression and/or activity (e.g., a small moleculeinhibitor, an RNAi agent or gene editing agent specific for ClpP) isadministered systemically, and in some cases locally (e.g., to theliver, e.g., see U.S. Pat. Nos. 8,524,679; and 8,008,468, both of whichare incorporated by reference in their entirety).

In some cases, the effect of an agent as described herein issubstantially liver-specific (e.g., the genome editing nuclease such asCre recombinase, a CRISPR/Cas endonuclease, a ZFN, a TALEN, etc., can bedelivered in a tissue specific manner or can be under the control of atissue specific promoter). By “substantially liver-specific” it is meantthat the majority of the reduction of ClpP expression is in the liver.Such can be achieved in a number of different ways. It is understoodthat the reduction of ClpP expression may not be limited only to theliver, but that one or more other tissues may exhibit reduction of ClpPexpression as well. For example, if a promoter is used (e.g., as part ofan expression vector to drive expression of a ClpP inhibitor, e.g., agenome editing nuclease, or a component of a ClpP inhibitor, e.g., aCRISPR/Cas guide RNA), that promoter need not be perfectly limited onlyto the liver. Such a promoter may exhibit ‘leaky’ expression in othertissues or may be limited to tissues other than just the liver (e.g.,the liver plus one or more additional tissues, but not all tissues,i.e., the promoter is not constitutive). As another example, if an agentis locally delivered (e.g., via direct injection), it is understood thatsome of the agent may also affect tissues in addition to the liver(e.g., neighboring organs, the blood, etc.). Thus, when the term“substantially liver-specific” is used, it is meant that global ClpPexpression (e.g., throughout the whole body) is not reduced, while liverClpP expression is reduced.

For example, when the agent is an RNAi agent, liver-specificity can beaccomplished by delivering the agent directly to the liver (e.g.,injection into the liver etc.). On the other hand, liver-specificity canbe accomplished by expressing the RNAi agent from a DNA encoding theagent (e.g., an expression vector encoding an shRNA) where the promoterdriving expression of the agent drives expression of the agent in theliver (e.g., a liver-specific promoter). Likewise, genome editing agentscan be delivered directly to the liver, or can be functional in theliver (e.g., by expressing one or more of the components from anexpression vector that has a promoter that drives expression in theliver, e.g., a liver-specific promoter). In some cases, the inhibitor ofClpP is administered such that reduction of ClpP expression issubstantially liver-specific, and the amount administered is effectivefor increasing insulin sensitivity of the individual.

Liver-specificity can also be accomplished when the inhibitor of ClpPthat reduces ClpP expression and/or activity is in an inactive formunless converted to an active form by a liver-specific enzyme. Forexample, the inhibitor of ClpP can be delivered in the form of aprodrug, which is then converted to an active form in the liver, forexample by a carboxylesterase such as human liver carboxylesterase 1(hCE1), a paraoxonase such as PON3, an alkaline phosphatase, humanvalacyclovirase (VACVase), a purine-nucleosidephosphorylase (PNP), andthe like). See, e.g., Yang, et. al., Enzyme-mediated hydrolyticactivation of prodrugs, Acta PharmaceuticaSinica B 2011; 1(3):143-159.

Agents can be prepared as injectables, either as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection can also be prepared. The preparationalso can be emulsified or encapsulated in liposomes or micro particlessuch as polylactide, polyglycolide, or copolymer for enhanced adjuvanteffect, as discussed above, Langer, Science 249: 1527, 1990 and Hanes,Advanced Drug Delivery Reviews 28: 97-119, 1997. Subject agents can beadministered in the form of a depot injection or implant preparationwhich can be formulated in such a manner as to permit a sustained orpulsatile release of the active ingredient. The agents can be formulatedas sterile, substantially isotonic and are in full compliance with allGood Manufacturing Practice (GMP) regulations of the U.S. Food and DrugAdministration.

Also within the scope of the disclosure are kits. For example, in somecases a subject kit can include (i) a detection reagent for detecting aClpP activity level and/or expression level (e.g., protein, mRNA), e.g.,an anti-ClpP antibody, and (ii) a control agent (e.g. a positive controlagent that is known to reduce a ClpP activity level and/or expressionlevel, and/or a negative control agent that is known not to reduce aClpP activity level and/or expression level) In some cases, a subjectkit can include instructions for use. Kits typically include a labelindicating the intended use of the contents of the kit. The term labelincludes any writing, or recorded material supplied on or with the kit,or which otherwise accompanies the kit.

Exemplary Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter describedabove may be beneficial alone or in combination, with one or more otheraspects or embodiments. Without limiting the foregoing description,certain non-limiting aspects of the disclosure numbered 1-26 areprovided below. As will be apparent to those of skill in the art uponreading this disclosure, each of the individually numbered aspects maybe used or combined with any of the preceding or following individuallynumbered aspects. This is intended to provide support for all suchcombinations of aspects and is not limited to combinations of aspectsexplicitly provided below:

1. A method of identifying a candidate agent for treating obesity, liverdisease, and/or diabetes, the method comprising:

-   -   (a) contacting a mammalian cell with a test agent;    -   (b) measuring the expression level and/or activity level of ClpP        in the mammalian cell relative to a reference value following        the contacting;    -   (c) determining that the test agent caused a decrease in the        expression level and/or activity level relative to the reference        value; and    -   (d) identifying the test agent as a candidate agent for treating        obesity, liver disease, and/or diabetes.        2. The method according to 1, wherein the mammalian cell is a        mouse cell.        3. The method according to 1, wherein the mammalian cell is a        human cell.        4. The method according to any of 1-3, wherein the mammalian        cell is in vitro.        5. The method according to any of 1-3, wherein the mammalian        cell is ex: vivo.        6. The method according to any of 1-3, wherein the mammalian        cell is in vim.        7. The method according to any of 1-6, wherein the mammalian        cell is a hepatocyte.        8. The method according to 2, wherein the contacting comprises        administering the test agent to a mouse.        9. The method according to 8, further comprising, measuring an        expression level and/or activity level of ClpP in the mouse        prior to the contacting of step (a) in order to obtain the        reference value.        10. The method according to any of 1-9, further comprising,        after step (d), administering the identified candidate agent to        an individual that has obesity, liver disease, and/or diabetes.        11. The method according to 10, wherein the individual is a        mouse, a non-human primate, or a human.        12. The method according to 10 or 11, further comprising, after        administering the identified candidate agent to the individual,        measuring one or more features of the individual selected from:        insulin sensitivity, blood glucose level, glucose tolerance,        body fat mass, an amount of fat tissue, an amount of white        adipose tissue; percent fat mass, body weight, visceral adipose        adipocyte size, plasma leptin level, growth hormone level, basal        energy expenditure, a level of phosphorylated AKT (p-AKT) in        muscles and/or fibroblasts, percent lean mass, mitochondrial        number in hepatocytes, mitochondrial mass in hepatocytes,        mitochondrial morphology in hepatocytes, fibroblast respiratory        capacity, fibroblast maximal oxygen consumption rate (OCR), and        fibroblast resistance to H₂O₂-induced cytotoxicity.        13. The method according to any of 1-12, wherein in the test        agent is a small molecule or a polypeptide.        14. The method according to any of 1-13, comprising measuring an        expression level of ClpP, wherein the expression level is an RNA        expression level and the measuring comprises the use of        quantitative RT-PCR, a microarray, or RNA sequencing.        15. The method according to any of 1-13, comprising measuring an        expression level of ClpP, wherein the expression level is a        protein expression level and the measuring comprises detecting        ClpP protein using an anti-ClpP antibody, mass spectrometry,        and/or an enzyme-linked immunosorbent assay (ELISA) assay.        16. The method according to any of 1-13, comprising measuring a        decrease in an activity level of ClpP by measuring an increase        in an amount of one or more proteins selected from: TNF        receptor-associated protein 1 (TRAP1), heat shock protein family        A (Hsp70) member 9 (Grp75), leucine rich pentatricopeptide        repeat containing (LRPPRC), caseinolytic mitochondrial matrix        peptidase chaperone subunit (ClpX), ornithine aminotransferase        (OAT), and Ion peptidase 1 (LonP1).        17. The method according to any of 1-16, wherein the method        comprises screening a plurality of test agents to identify one        or more candidate agents for treating obesity, liver disease,        and/or diabetes.        18. A method of treating an individual with obesity, liver        disease, and/or diabetes, the method comprising:    -   administering an inhibitor of ClpP to the individual in an        amount effective for decreasing an amount of fat tissue in the        individual, preventing or reducing weight gain of the        individual, increasing insulin sensitivity of the individual,        and/or increasing glucose tolerance of the individual.        19. The method according to 18, wherein the inhibitor of ClpP is        an RNAi agent or a gene editing agent that specifically reduces        expression of ClpP.        20. The method according to 18 or 19, wherein the inhibitor of        ClpP is administered such that reduction of ClpP expression is        substantially liver-specific, and the amount administered is        effective for increasing insulin sensitivity of the individual.        21. The method according to 18, wherein the inhibitor of ClpP is        a small molecule.        22. The method according to 21, wherein the small molecule is a        13-Lactone.        23. The method according to 22, wherein the β-Lactone is        (3RS,4RS)-3-(pon-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one        or a pharmaceutically acceptable salt thereof.        24. The method according to any of 18-23, wherein the inhibitor        of ClpP is delivered directly to the individual's liver, and the        amount administered is effective for increasing insulin        sensitivity of the individual.        25. The method according to any of 18-24, wherein the        administering comprises local injection.        26. The method according to any of 18-25, further comprising a        step of measuring insulin sensitivity of the individual.

It will be apparent to one of ordinary skill in the art that variouschanges and modifications can be made without departing from the spiritor scope of the invention.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of the invention nor are they intended to represent that theexperiments below are all or the only experiments performed. Effortshave been made to ensure accuracy with respect to numbers used amounts,temperature, etc) but some experimental errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference.

The present invention has been described in terms of particularembodiments found or proposed to comprise preferred modes for thepractice of the invention. It will be appreciated by those of skill inthe art that, in light of the present disclosure, numerous modificationsand changes can be made in the particular embodiments exemplifiedwithout departing from the intended scope of the invention. For example,due to codon redundancy, changes can be made in the underlying DNAsequence without affecting the protein sequence. Moreover, due tobiological functional equivalency considerations, changes can be made inprotein structure without affecting the biological action in kind oramount. All such modifications are intended to be included within thescope of the appended claims.

Materials and Methods

The following methods were utilized in the examples described herein:

Animals. All mice were maintained on a 12-h light/dark cycle in apathogen-free animal facility. Mice were separately housed by theirgenotypes for all metabolic studies. For high-fat diet (HFD)-inducedobesity studies, animals were fed a FWD (21% by weight, 42% kcal fromfat, 0.2% total cholesterol; TD.01064, Harlan-Teklad) for specifiedperiods. Anesthesia was induced with isoflurane (survival procedures) orwith avertin (terminal procedures). All procedures were approved by theUCSF Animal Research Committee and followed NIH guidelines. Animalsample sizes for different assays were chosen based on previousliterature. Animals were grouped according to genotype; no randomizationwas used. For all behavior tests, all animal Ins were blinded to tester,Testers were not blinded for other assays.

Generation and maintenance of ClpP knockout (ClpP^(−/−)) and ClpPconditional knockout (ClpP-cKO) mice. Frozen ClpP^(+/−) sperm wasrecovered and ClpP^(+/−) mice were generated. ClpP^(−/−) mice wereback-crossed into a C57BL/6 genetic background for three or moregenerations. Since Clp^(−/−) mice were infertile, ClpP^(+/−) mice werebred with each other to generate littermates with three differentgenotypes (WT, ClpP^(+/−), ClpP^(−/−)) and used to maintain the line.

Generation and maintenance of ClpP conditional knockout (Clp-cKO) mice.A ClpP-cKO construct was made by flanking ClpP gene with a long arm ofhomology (5′ terminal, about 6 Kb) and a short arm of homology (3′terminal, about 3 Kb). LoxP sites were added to both ends of the regioncontaining ClpP exons 1-3 for future removal. A puromycin resistant geneflanked by F3 sites were inserted right before the 3′ terminal loxPsite. A mouse ES cell clone carrying the targeted ClpP allele wasselected and used for microinjection. Resulting chimeras were bred withFlp mice to generate offspring carrying the targeted ClpP allele.Heterozygous ClpP-cKO mice were then bred to generate homozygousClpP-cKO mice. The ClpP-cKO mice were generated on a C57BL/6 geneticbackground. Homozygous ClpP-cKO mice were used to maintain the line.

Body composition analysis. Body composition of mice after a 4-h fast wasanalyzed under isoflurane anesthesia by dual-energy X-ray absorptiometry(DEXA) with a PIXImus2 scanner (GE Healthcare Lunar).

Food intake study. Mice were separately housed by their genotypes. Foreach cage, food was weighted every day at 10:00 a.m. for 4 days. Thedaily food intake for each cage was calculated by subtracting the foodweight at the end of a 24-h period from the food weight at the startingtime. The daily food intake is normalized either animal number or bodyweight.

Fasting-refeeding study. Mice fasted for 24 h and then returned to freefeeding. The body weights of mice were measured before and after thefasting. The food intake and body weight gain were monitored afterrefeeding periods of 8 and 24 h.

Body temperature measurement. The core body temperatures of mice weretaken with a rodent rectal thermometer.

Measurement of blood glucose and plasma insulin. Blood glucose wasmeasured with a glucometer and glucose test strips (Free Style Lite).Plasma insulin was quantified with an insulin Elisa kit (Crystal Chem).

Glucose, insulin, and pyruvate tolerance tests, Glucose, insulin, andpyruvate tolerance tests were performed by intraperitoneal injection ofglucose (2 g/kg), insulin (0.5 units/kg) or pyruvate (2 g/kg) after anovernight fast for glucose and pyruvate or a 4-h fast for insulin. Bloodglucose levels were measured before injection and at different timepoints after injection. To assess glucose-stimulated insulin release,mice fasted overnight (for 16 h) and were then injectedintraperitoneally with glucose (2 g/kg). Blood samples were collectedfrom tail veins before injection and at different time points afterinjection.

Behavior tests. The general neurological behavior profiles of ClpP^(−/−)mice were assessed by the grip strength, incline, and tail suspensetests. The motor function of these mice was determined by the rotarodtest. Anxiety levels were determined by elevated plus maze. Activitylevels were studied by an open field test. The learning and memory ofthese mice were examined using the Morris water maze. The auditoryfunctions were tested by prepulse inhibition test.

Histological analysis. Tissues (liver, visceral adipose, gastrocnemiusmuscle, interscapular brown adipose, and pancreas) were fixed in 4%paraformaldehyde (pH 7.4) overnight, embedded in paraffin, and seriallysectioned at 7-8 μm. Standard haematoxylin and eosin staining wasperformed.

Adipocyte size measurement, Haematoxylin and eosin staining of adiposetissue sections was performed and images were analyzed with image J.

Electron microscopic analysis of liver sections, WT or ClpP^(−/−) micewere perfused with EM fixative. The liver tissues were collected andfixed in the EM fixative for 2 days. The tissues were processed andelectron microscopy images were collected using a JEOL, JEM-1230transmission electron microscope. The mitochondrial number, area, androundness were measured by Image J software at 13,600 magnifications.

Generation of adipocyte-specific ClpP-cKO mice. Adipocyte-specific ClpPknockout mice were generated through breeding ClpP-cKO mice with ap2-Cremice. The Cre⁺ mice had greatly reduced ClpP protein levels in adiposetissues and the Cre-littermates were served as controls in metabolicassays.

Tail vein injection of AAV-CMV-Cre. AAV8.2-CMV-Cre and control AAV8.2were utilized. These viruses were injected into 3-5 month-old ClpP-cKOmice through tail vein at a dose of 5×10⁹ genome copies/gram (gc/gram)body weight. The body weights of the injected mice were measured onceevery week. The blood glucose and plasma insulin levels were measuredbefore and 3 weeks after injection. The glucose tolerance test wasperformed on these mice 4 weeks after injection. HFD was given to thesemice 6 weeks after injection. The body weight gain in response to HFDwas followed and the glucose tolerance test was performed after twoweeks on HFD.

Western blot, Tissues or cells were lysated in low-detergent buffer (50mM Tris/HCl, pH 8.0, 150 call NaCl, 0.1% SDS, 0.5% Nonidet P-40, 0.5%sodium deoxycholate) with a mixture of protease inhibitors. Phosphataseinhibitors were also included in the lysate buffer. The lysates werecentrifuged at 13,000 rpm for 10 min to remove the pellets. SDS-PAGE wasperformed with a WAGE his-tris system from Invitrogen, following thevendor's protocol. A standard western blot protocol was used withIRDye-labeled secondary antibodies (Li-cor). A Li-cor image system wasused to scan the western blot images.

2D fluorescence difference gel electrophoresis. Organs from ClpP^(−/−),ClpP^(+/−), or WT mice were submitted for 2D fluorescence difference gelelectrophoresis analysis. The most dramatically changed spots were cutfrom the gel for mass spectrometry analysis to identify the proteins,which was done by the same company.

Mouse fibroblast preparation. Mouse fibroblasts were prepared from skintissue collected from new-born pups. The tissue was cut into smallpieces and placed on a 100-mm cell culture dish with the skirt side up.After 5-10 min, 10 ml of fibroblast medium (DMEM with 10% FBS) wasslowly added to the dishes. The implants were cultured in a humidified37° C., 5% CO₂ incubator. Fibroblasts eventually proliferated after 7-10day in culture. Mouse fibroblasts were maintained in the same medium formultiple passages.

Overexpression of ClpP in ClpP^(−/−) fibroblasts. Lentiviraloverexpression vector for human ClpP was constructed with pLenti7.3TOPOTA Cloning Kit from Life Technologies. The lentiviruses were packed withViraPower™ lentiviral packaging mix from Life Technologies. Lentiviruscarrying either empty vector (control) or human ClpP cDNA were added toClpP−/− fibroblasts and incubated for 48 hr before further experiments.

shRNA knockdown of different genes in ClpP^(−/−) fibroblasts. LentiviralshRNA constructs were obtained. The lentiviruses were packed withViraPower™ lentiviral packaging mix from Life Technologies. Lentiviruscarrying either empty vector (control) or different shRNAs were added toClpP^(−/−) fibroblasts and incubated for 48 hr before furtherexperiments.

Cell viability assay. Mouse fibroblasts were plated on blackclear-bottomed 96-well plates at 10,000 cells per well. After incubatingfor 24 h, the cells were cultured in OPTI-MEM medium overnight. Thecells were treated with H₂O₂ at various doses for 4 h. Alamar bluereagent (Invitrogen) was then added to the wells (1:10 ratio). After a2-h incubation, the fluorescence intensity was measured at 590 nmemission (560 nm excitation).

Seahorse OCR assay. Mouse fibroblasts were maintained in fibroblastgrowth medium until confluence. The day before the assay, fibroblastswere trypsinized and plated on a 96-well Seahorse culture plate at 40000cells per well. Before the assay, the culture medium was changed to aCO₂-independent medium supplied by Seahorse Bioscience. The cells werethen incubated at 37° C. without CO₂ for 0.5-1 h. The oxygen consumptionrate (OCR) assay was performed with an NT Cell mito-stress test kit andXF Extracellular Flux Analyzers (Seahorse Bioscience) following thevendor's protocol.

Growth hormone (Gil) and leptin level determination. The GH and leptinlevels in mouse plasma were determine by a mouse GH ELISA kit and ultrasensitive mouse insulin ELISA kit from Crystal Chem, Inc.

Antibodies. The antibodies used were anti-ClpP (rabbit, NovusBiologicals), anti-ClpX (SDI, custom made), anti-Akt (rabbit, CellSignaling), anti-phospho-Akt (rabbit, Cell Signaling), anti-Grp75(rabbit, Cell Signaling), anti-TRAP1 (moue mAb-AbCam), anti-LRPPRC(rabbit, Proteintech Group), anti-LonP (rabbit, Sigma-Aldrich), anti-OAT(mouse, Abeam), anti-SDH2 (rabbit, aric antibodies), anti-ATP6V1A(rabbit, Proteintech Group), anti-CPS1 (mouse, Lifespan), anti-Hsp70(rabbit, Cell Signaling), anti-GAPDH (mouse mAb, Millipore), anti Hsp60(rabbit, Abeam), anti-VDAC (mouse mAb, EMD Chemical), and anti-actin(rabbit, Sigma-Aldrich).

Statistical analyses. Data are presented as mean±SD unless otherwisespecified. All statistical analyses were done using Prism 6 software(GraphPad). Differences between means were assessed by t-test, one-wayANOVA, or repeated measures ANOVA, followed by Bonferroni orTukey-Kramer post hoc tests. In all cases, P<0.05 was consideredstatistically significant.

Mammalian ClpXP, an ATPase complex consisting of the catalytic subunitClpP and the regulatory subunit ClpX, is a mitochondrial protease withunclear physiological functions. The data presented in the followingexamples show that ClpP knockout (ClpP^(−/−)) mice expended more energyand had reduced adipose tissue and enhanced insulin sensitivity comparedto wild type controls. Drastic increases in mitochondrial chaperoneswere detected in various organs of ClpP^(−/−) mice, accompanied withincreased mitochondrial numbers. Eliminating ClpP increasedmitochondrial function and anti-stress capacity of cells by elevatingLRPPRC and/or TRAP1 levels (both of which are proteins with roles inmitochondrial function), which were reversed by knocking down eitherprotein. Hepatic ClpP was responsible for regulating insulinsensitivity, while adipocytic ClpP was not, Thus, ClpP is a masterregulator of mitochondrial function and stress response, which in turnmodulate energy homeostasis, lipid storage, and insulin sensitivity.These data highlight ClpP as a therapeutic target for treating obesityand diabetes.

Example 1: Absence of ClpP in Mice Reduced Adipose Tissue on a Chow orHigh Fat Diet

To understand the physiological roles of mammalian ClpXP protease, aClpP knockout (ClpP^(−/−)) mouse model was generated through agene-trapping strategy (FIG. 7, panel a). Quantitative polymerase chainreaction (qPCR) (not shown) and western blotting confirmed that the Clppgene was efficiently deleted in different organs (FIG. 7, panels h-i).ClpP^(−/−) pups were born viable and at normal Mendelian ratios.Morphological assessment of liver and muscle revealed no abnormalitiesin 6-month-old homozygous (ClpP^(−/−)) or heterozygous (ClpP^(+/−))knockout mice (FIG. 8). There were no differences between ClpP^(−/−) andWT littermates in general neurological behavior, motor function (rotarodand swim speed tests), anxiety (elevated plus maze test), or learningand memory (Morris water maze test) (FIG. 9), indicating a normalneurological profile.

However, both male and female adult ClpP^(−/−) mice were significantlysmaller in weight and size than WT and ClpP^(+/−) littermates (FIG. 1,panels a-b). In contrast, all new-born pups had similar body weightsregardless of their ClpP genotypes (FIG. 1, panel c). In addition,growth hormone levels in 5-6-month-old ClpP^(−/−) mice were higher thanin other groups of mice (ClpP^(−/−), 8.54±5.41 ng/ml; ClpP^(+/−),2.48±0.67 ng/ml; WT, 0.91±0.53 ng/ml; n=3-5 mice for each genotype; oneway ANOVA, p=0.0053), so body weight differences in adults did not stemfrom early developmental defects or growth hormone deficiencies.

Clp^(−/−) mice had dramatically lower body fat, as measured by totalbody fat mass or body fat content (% fat mass) (FIG. 1, panels d-e),than WT mice. Although ClpP^(+/−) mice had similar body weight to WTmice, they tended to have lower body fat (FIG. 1, panels d-e). Incontrast, the lean content (% lean mass) of ClpP^(−/−) mice wassignificantly higher than that of ClpP^(+/−) and WT mice (FIG. 1, panelg), although the lean mass was significantly lower (FIG. 1, panel f).Interestingly, quantification of interscapular brown adipose tissue,which is critical in thermogenesis and beneficial in metabolicregulation, showed a similar profile as lean tissue among three ClpPgenotypes (FIG. 1, panels h-i). Consistent with their lower body fatcontent, visceral adipose from ClpP^(−/−) mice had smaller adipocytescompared to those from ClpP^(+/−) and WT mice (FIG. 1, panel m, and FIG.10), in contrast, the morphology of brown adipose tissue in ClpP^(−/−),ClpP^(+/−), and WT mice were similar (FIG. 11, panel a). Thus, knockingout ClpP greatly reduces white adipose tissue, which likely contributesto the lower body weights seen in adult ClpP^(−/−) mice.

Next, mice were fed a high-fat diet (HFD) for 2 months to determine theeffect of eliminating ClpP on HFD-induced obesity. ClpP^(−/−) mice wereresistant to HFD-induced weight gain, and ClpP^(+/−) mice showed delayedweight gain within the first 10 days (FIG. 1, panel j, and FIG. 12,panel a). At 40 days on the HFD, when body weight changes in all threegroups had plateaued, ClpP^(−/−) mice showed only a small increase infat mass and body weight, while ClpP^(+/−) and WT mice gainedsignificantly more (FIG. 1, panel k, FIG. 1, panel l). Thus, ClpP^(−/−)mice are resistant to HFD-induced obesity, HFD did not change thedifferences in lean mass and content among various groups of mice(comparing FIG. 1, panels f-g, with FIG. 12, panels c-d).

Also consistent with lower body fat content, ClpP^(−/−) mice had muchlower levels of plasma leptin than other groups of mice (ClpP^(−/−),0.409±0.224 ng/ml; ClpP^(+/−), 1.927±1.928 minis; WT, 2.056±2.022;n=11-12 mice at 5-6 months of age, one way ANOVA, p<0.05).Interestingly, ClpP^(−/−) mice were infertile, as observed in leptindepleted animal models. While not intending to be bound by anyparticular theory, it is possible that low leptin levels are responsiblefor the reproductive defect in ClpP^(−/−) mice.

Example 2: Absence of ClpP in Mice Altered the Enemy ExpenditureProfiles

Low leptin levels lead to feeding behavior and adipose deposition in WTmice. Consistent with lower serum leptin levels, the food intakenormalized to body weight was significantly higher in ClpP^(−/−) micethan in WT and Clp^(+/−) mice (FIG. 2, panel b), although mice in allgroups had similar food intake per animal per day (FIG. 2, panel a).Thus, ClpP^(−/−) mice gained less weight and generated less fat whileconsuming more food, suggesting that they either expended more energy ordid not utilize nutrition properly.

A fasting-refeeding approach was used to dissect these two possibilities(FIG. 2, panel c). After 24 h of fasting, ClpP^(−/−) mice lost the sameamount of weight as WT and ClpP^(+/−) mice (FIG. 2, panel d). SinceClpP^(−/−) mice had lower body weight, their percentage of body weightloss was significantly higher than that of WT and ClpP^(+/−) formates(FIG. 2, panel e), suggesting that ClpP^(−/−) mice expend more energy.During the refeeding period, ClpP^(−/−) mice consumed significantly morefood and gained a significantly higher percentage of weight comparedwith WT and ClpP^(+/−) littermates (FIG. 2, panels f-g).

Higher energy consumption may be attributable to hyperactivity or highthermogenesis.

The open field test showed similar activity levels among all groups(FIG. 13). Surprisingly, the core body temperature was significantlylower in ClpP^(−/−) mice than in WT and ClpP^(+/−) mice (FIG. 2, panelsh-i), suggesting that ClpP^(−/−) mice did not use more energy forthermogenesis. Thus, it is likely that the ClpP^(−/−) mice had higherbasal energy expenditure.

Example 3: Absence of ClpP in Mice Improved Insulin Sensitivity

Blood glucose levels and plasma insulin levels in ClpP^(−/−) mice wereanalyzed. Blood glucose levels were significantly lower in ClpP^(−/−)mice than in ClpP^(+/−) and WT mice (FIG. 3, panel a). Plasma insulinlevels were also significantly lower in ClpP^(−/−) mice than in WTcontrols, with those in ClpP^(+/−) mice being in the middle, under bothfree-feeding and fasting conditions (FIG. 3, panels b-c). The lowinsulin levels were unlikely due to a pancreas deficit, based on themorphological data (FIG. 11, panel b). Thus, ClpP^(−/−) mice maintainednormal blood glucose levels in spite of having very low insulin levels.After 2 months on HFD, blood glucose levels in WT and ClpP^(+/−) miceincreased greatly. In contrast, ClpP^(−/−) mice maintained much lowerblood glucose and insulin levels on HFD (FIG. 3, panels d-e). Glucoseand insulin tolerance tests further revealed enhanced insulinsensitivity in ClpP^(−/−) mice (FIG. 3, panels f-h). A trend towardincreased pyruvate tolerance in ClpP^(−/−) mice was detected, althoughnot reaching significance (FIG. 3, panel i). In support of increasedinsulin sensitivity, higher p-AKT levels, a main component of theinsulin signaling pathway, were found in muscles and fibroblasts ofClpP^(−/−) mice (FIG. 3, panels j-l). Additionally, ClpP^(−/−)fibroblasts were more sensitive than WT fibroblasts to IGF-induced AKTactivation in culture (FIG. 3, panel m). These data strongly support theconclusion that eliminating ClpP improves insulin sensitivity in mice.

ClpP^(−/−) mice were cross-bred with db/db mice (a model of obesity,diabetes, and dyslipidemia) to determine whether and how depleting ClpPaffects the obese and diabetic phenotypes of db/db mice. Knocking outClpP led to a small but significant decrease in body weight of db/dhmice (FIG. 14, panel a). ClpP knockout also significantly decreasedblood glucose levels in db/db mice after 4 h of fasting (FIG. 3, paneln), although this effect disappeared after 16 h of fasting probably dueto the fact that the glucose levels in db/db mice with WT ClpP after 16h fasting were already dropped to normal levels (FIG. 14, panels b-c).More importantly, eliminating ClpP greatly improved the glucosetolerance of db/db mice (FIG. 3, panel o).

Example 4: Absence of ClpP in Mice Upregulates Mitochondrial ChaperonLevels

To identify downstream effectors of ClpP, two dimensional (2D)fluorescence difference gel electrophoresis (2D-DICE) was employed tocompare the protein profiles of various organs from mice with differentClpP genotypes (FIG. 15). Mass spectrometry was then used to identifythe most dramatically changed proteins in different organs of ClpP^(−/−)mice (FIG. 20). Validation by western blots confirmed six potentialClpXP downstream effectors (TRAP1, Grp75, LRPPRC, ClpX, OAT, and LonP;FIG. 21); their protein levels increased greatly in multiple organs(FIG. 4, panels a-h and FIG. 16), but their mRNA levels remained thesame in most cases (FIG. 22). Thus, ClpP likely controls the levels ofthese proteins post-transcriptionally. Significantly increased proteinlevels were also detected in embryonic day-19 pups (not shown), withouta decrease in body weight, suggesting that the accumulation of theseproteins is likely the cause, rather than a consequence, of the observedphenotypes. Increased levels of the same proteins were also detected inmouse fibroblasts (FIG. 4, panels i-j), Which were subsequently reducedby overexpressing mouse ClpP (FIG. 4, panel k-l), suggesting that ClpPspecifically regulates these protein levels in a cell-autonomous manner.

Strikingly, many of the potential ClpP downstream effectors are relatedto mitochondrial protein homeostasis, including the molecular chaperonesClpX (mitochondrial Hsp100), TRAP1 (mitochondrial Hsp90), and Grp75(mitochondrial Hsp70) as well as mitochondrial protease LonP (FIG. 4,panels a-h, FIG. 16). ClpP knockout had no significant effect on thelevels of mitochondrial proteins VDAC and Hsp60 (FIG. 16). Furthermore,there were no significant differences in many cytosol and endoplasmicreticulum (ER) heat shock proteins, such as Bip and Hsp90 (FIG. 16).Thus, either directly or indirectly, ClpP controls a specific group ofmitochondrial proteins, especially chaperons.

Example 5: Absence of ClpP Increased Mitochondrial Numbers and EnhancedMitochondrial Function and Anti-Stress Capacity

Mitochondrial number and function were analyzed in ClpP^(−/−) mice.Electron microscopic (EM) study of ClpP^(−/−) liver sections showed thatdepletion of ClpP increased mitochondrial numbers and alteredmitochondrial morphology in hepatocytes (FIG. 5, panels a-b and FIG. 17,panels a-b), Mitochondrial mass (measured by total mitochondrial areaper field) was also significantly increased in ClpP^(−/−) hepatocytes(FIG. 5, panel c, and FIG. 17, panels a-b). The ClpP^(−/−) mitochondriahad denser matrixes compared to those of WT (FIG. 5, panel a, and FIG.17, panels a-b), probably due to increased protein content in ClpP^(−/−)mitochondria. Although the size distribution of mitochondria was similarbetween ClpP^(−/−) and WT hepatocytes (FIG. 17, panel c), there was ahigher percentage of ClpP^(−/−) mitochondria with lower roundness (FIG.17, panel d), suggesting that more ClpP^(−/−) mitochondria had elongatedshapes.

To determine whether ClpP regulates mitochondrial function, therespiratory capacity of fibroblasts from ClpP^(−/−) and WT mice werecompared using a Seahorse XF analyzer. ClpP^(−/−) fibroblasts had asimilar basal oxygen consumption rate (OCR) but a significantly highermaximal OCR compared with WT fibroblasts (FIG. 5, panel d), indicatingenhanced mitochondrial function in the absence of ClpP. ClpP^(−/−)fibroblasts also showed resistance to H₂O₂-induced cytotoxicity comparedwith WT fibroblasts (FIG. 5, panel e), suggesting increasedanti-oxidative stress capacity in the absence of ClpP. Overexpression ofmouse ClpP in ClpP^(−/−) fibroblasts not only led to decreased levels ofthe ClpP downstream effectors (FIG. 4, panels k-l) but also abolishedthe enhancement of mitochondrial function and resistance to H₂O₂-inducedcytotoxicity observed in ClpP^(−/−) cells (FIG. 5, panels f-g).

Example 6: Mitochondrial Effectors TRAP1, Grp75, and LRPPRC Mediated theEffects of ClpP Absence on Mitochondrial Function and Anti-StressCapacity

To determine whether and which of the mitochondrial effectors regulatedby ClpP mediated the enhanced mitochondrial function seen in ClpP^(−/−)fibroblasts, lentiviral shRNAs were used to knock down each of them inClpP^(−/−) fibroblasts. This was followed by functional assays (FIG. 18,panels a-d). Knocking down TRAP1 or Grp75, but not LRPPRC and ClpX,abolished or lowered the resistance of ClpP^(−/−) fibroblasts to H₂O₂cytotoxicity (FIG. 5, panels h-i and FIG. 18, panels e-f). Knocking downeither LRPPRC or TRAP1 abolished the enhanced respiratory capacity ofClpP^(−/−) cells, while lowering the level of ClpX or Grp75 had no suchan effect (FIG. 5, panel j). These data suggest that ClpP regulatesmitochondrial function by controlling its effector levels, specificallyTRAP1, Grp75, and LRPPRC. These proteins may affect mitochondrialfunction via distinct pathways or have synergistic effects on the samepathway.

Example 7: Hepatic ClpP was Responsible for Regulating InsulinSensitivity

Multiple tissues can affect insulin sensitivity through differentmechanisms. ClpP conditional knockout mice (ClpP-cKO) were employed tofurther dissect the tissue specific mechanism(s) underlying thephenotypes of ClpP^(−/−) mice. In this mouse model, LoxP sites wereinserted to flank the mouse genomic region containing ClpP exons 1-3 forfuture removal. To delete ClpP specifically in the liver, AAV-CMV-Crewas injected through the tail vein into ClpP-cKO mice. The ClpP levelsin livers, detected by immunostaining, were dramatically reduced inCre-injected mice compared to control AAV-injected mice (FIG. 6, panela). At three weeks after injection, the Cre-injected mice had asignificantly lower body weight gain than the control mice (FIG. 6,panel d), although the total body weights were not significantlydifferent between the two groups of mice (FIG. 6, panels b-c). The bloodglucose levels of the Cre-injected mice were also significantly lowerthan those of the control mice (FIG. 6, panel f), while no difference inplasma insulin levels was detected (FIG. 6, panel e), The enhancedinsulin sensitivity of Cre-injected mice was revealed by glucosetolerance test in mice on either chow diet or HFD for two weeks (FIG. 6,panels g-h). Unlike ClpP^(−/−) mice, liver specific knockdown of ClpPdid not affect FWD-induced body weight gain (FIG. 6, panel i), Thesedata suggest that hepatic ClpP levels are responsible for regulating theinsulin sensitivity observed in ClpP^(−/−) mice, without affecting theadipose content.

ClpP-cKO mice were also crossed with ap2-Cre mice to establishadipocyte-specific ClpP-KO mice. The ClpP levels in adipose tissues ofClpP-cKO/ap2-Cre mice were about a quarter of those in control mice(FIG. 19, panels a-b). It was not clear whether the residue ClpP levelswere due to uncompleted deletion of Clpp by apt-Cry; or contamination ofother tissues or cells (such as vascular or immune cells) in thecollected adipose tissues. No significant changes in body weights, bloodglucose levels, and glucose tolerance were detected in ClpP-ckO/ap2-Cremice (FIG. 19, panels c-e). The body weight gain and glucose toleranceof ClpP-cKO/ap2-Cre mice on HFD were also similar to those of controlmice on the same diet (FIG. 19, panels f-g). Therefore, adipocytic ClpPis unlikely attributable to the reduced adipose deposition and enhancedinsulin sensitivity seen in ClpP^(−/−) mice.

Example 8: In-Vitro Analysis of Small Molecule ClpP Inhibitors in MouseEmbryonic Fibroblast (MEF) Cells

Wild type mouse MEF cells were seeded at 200,000 cells per well at 6well plates the day before treatment. Cells were treated with the smallmolecule ClpP inhibitors A2-32-01, AV167, and AV179 (describedpreviously herein) at 10 μM in OPTI-MEM twice a day for 2 days (48 hr).For each treatment, the old medium will be removed and fresh mediumadded to the wells. After 48 hr treatment, the cells were harvested forwestern blot analysis or gPCR assay of ClpP substrates.

As shown in FIGS. 24 and 25, treatment with ClpP inhibitor A2-32-01increased ClpP substrate protein levels in mouse WEF cells, mimickingthe phenotypes observed in ClpP knock out MEF and in ClpP knock outmouse organs. As shown in FIG. 26, the mRNA level of these genes inmouse MEF cells remained unchanged after A2-32-01 treatment, whichexcluded potential off-target effects of A2-32-01 through transcriptionregulation.

Based on these in vitro data, further in vivo testing of A2-32-01 inhigh fat diet (HFD)-induced obesity and diabetes mouse models wasperformed as described in Example 9 below.

Example 9: In-Vivo Analysis of Small Molecule ClpP Inhibitor A2-32-01 inHigh Fat Diet (HFD)-Induced Obesity and Diabetes Mouse Models

7-9 month old WT female (20 mice), were fed with high fat diet for 3weeks. Body weight change and blood glucose/plasma insulin levels inresponse to HFD were measured. At the beginning of the 4^(th) week, fortreatment group, A2-32-01 was injected peritoneally twice daily at 300mg/kg for 8 days. For the control group, corn oil (vehicle) wasinjected. The mice were maintained on HFD. The body weight change wasfollowed every day. At the 8^(th) day of A2-32-01 injection, bloodglucose levels were measured, and a glucose tolerance test was appliedto measure insulin sensitivity. Body weight data from the first sevendays of in vivo A2-32-01 treatment is provided in FIG. 28, which show asignificant decrease in body weight on day 2 through day 7 as comparedto the vehicle control group. A glucose tolerance test, which reflectsinsulin sensitivity, was performed on day 8 and the data are provided inFIG. 29, which show a significant improvement of insulin sensitivity inA2-32-01-treated mice as compared to the vehicle control mice.

Example 10: Continued In-Vivo Analysis of Small Molecule ClpP InhibitorA2-32-01 in High Fat Diet (HFD)-Induced Obesity and Diabetes MouseModels (Prophetic)

The in-vivo analysis of Example 9 is continued, with collection of theblood and multiple organs (liver/muscle/brain) at day 9 of A2-32-01injection. Mitochondria are isolated from mouse liver and ClpP activityis tested using peptide substrates, e.g., as described herein. Westernblot analysis is performed to detect ClpP substrate levels.

Example 11: Continued In-Vivo Analysis of Small Molecule ClpP InhibitorA2-32-01 in High Fat Diet (HEM-Induced Obesity and Diabetes Mouse Models

The in-vivo analysis described in Examples 9 and 10 was continued inaccordance with the experimental protocol shown in FIG. 30. As shown inFIG. 31 (left panel), the A2-32-01-treated mice on HFD had significantlydecreased body weight compared to vehicle-treated mice on HFD. As shownin FIG. 31 (right panel), glucose tolerance tests showed increasedinsulin sensitivity in HFD-fed WT mice treated with A2-32-01 compared tothose treated with vehicle. ClpP activity was lower in livermitochondrion lysates from A2-32-01-treated mice compared to those fromvehicle-treated mice as shown in FIG. 32, left panel. As shown in FIG.32, right panel, the levels of tentative ClpP effectors Trap1, ClpX,LRPPRC, LonP, and OAT), measured by western blot, were higher in livermitochondrion lysates from A2-32-01-treated mice compared to those fromvehicle-treated mice. Finally, FIG. 33 shows liver morphology fromvehicle or A2-32-01-treated WT mice on HFD, indicating that A2-32-01treatment lowered the lipid accumulation in liver cells and restorednormal morphology of liver cells in WT mice on HID, as compared tovehicle-treated WT mice on HID. Overall, these data demonstrate thattreatment with the ClpP inhibitor A2-32-01 in high fat diet (HFD)-fedwildtype (WT) mice significantly lowers body weight, increases insulinsensitivity, and restores normal liver morphology.

Example 12: In-Vivo Analysis of Small Molecule ClpP Inhibitor A2-32-01in db/db Mice

The following in-vivo analysis of db/db mice (a model of obesity,diabetes, and dyslipidemia—see, e.g., Kobayashi et al. Metabolism, 2000January; 49(1):22-31.) was conducted according to the experimentalscheme set forth in FIG. 34, panel A. As shown in FIG. 34, panel B, theA2-32-01-treated db/db mice had significantly decreased body weightcompared to vehicle-treated dh/db mice at the end of the treatment. Inaddition, glucose tolerance tests showed increased insulin sensitivityin db/db mice treated with A2-32-01 compared with those treated withvehicle (FIG. 35, panel C). Fasting blood glucose levels in db; db micetreated with A2-32-01 were significantly lower than those treated withvehicle as shown in FIG. 35, panel D. ClpP activity was lower in livermitochondria lysates from db/db mice treated with A2-32-01 compared tothose treated with vehicle as shown in FIG. 35, panel E. FIG. 36, panelsF-I, show liver morphology from vehicle or A2-32-01-treated db/db mice,indicating that A2-32-01 treatment lowered the lipid accumulation inliver cells and restored normal morphology of liver cells in db/db mice,as compared to vehicle-treated db/db mice. Overall, these datademonstrate that treatment with ClpP inhibitor A2-32-01 in db/db micesignificantly lowers body weight, increases insulin sensitivity, andrestores normal liver morphology.

What is claimed is:
 1. A method of treating an individual with obesity,liver disease, and/or diabetes, the method comprising: administering aninhibitor of ClpP to the individual in an amount effective fordecreasing an amount of fat tissue in the individual, preventing orreducing weight gain of the individual, increasing insulin sensitivityof the individual, and/or increasing glucose tolerance of theindividual.
 2. The method according to claim 1, wherein the inhibitor ofClpP is an RNAi agent or a gene editing agent that specifically reducesexpression of ClpP.
 3. The method according to claim 1, wherein theinhibitor of ClpP is administered such that reduction of ClpP expressionis substantially liver-specific, and the amount administered iseffective for increasing insulin sensitivity of the individual.
 4. Themethod according to claim 1, wherein the inhibitor of ClpP is a smallmolecule.
 5. The method according to claim 4, wherein the small moleculeis a β-Lactone.
 6. The method according to claim 5, wherein theβ-Lactone is(3RS,4RS)-3-(non-8-en-1-yl)-4-(2-(pyridin-3-yl)ethyl)oxetan-2-one or apharmaceutically acceptable salt thereof.
 7. The method according toclaim 1, wherein the inhibitor of ClpP is delivered directly to theindividual's liver, and the amount administered is effective forincreasing insulin sensitivity of the individual.
 8. The methodaccording to claim 1, wherein the administering comprises localinjection.
 9. The method according to claim 1, further comprising a stepof measuring insulin sensitivity of the individual.